L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

Possible Host Adaptation as an Evolution Factor of Cowpea aphid‐borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid‐borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C. hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous‐infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea‐infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.     

Anatomical and physiological changes of in vitro-propagated Vriesea imperialis (Bromeliaceae) in the function of sucrose and ventilated containers

Abstract

Under in vitro culture conditions, plants may present physiological and anatomical disorders, which can interfere negatively after ex vitro transfer. The aim of this investigation was to analyze the impacts of natural ventilation and sucrose supply on the anatomy and physiology of Vriesea imperialis. Plants previously grown in vitro were transferred to culture medium containing 0, 15, 30 or 45?g L^?1 sucrose. Three different culture container sealing systems were tested: lids with a green filter (81.35 gas exchanges per day), yellow filter (13.09 gas exchanges per day) or lids with a yellow filter covered with three layers of transparent polyvinylchloride (PVC) film (blocking fluent gas exchange). Sucrose concentrations influenced thickness, lignin and suberin deposition of exodermis cell wall. The modifications verified in leaves, such as higher density of stomata and trichome scales, showed that sucrose can induce osmotic stress in the plants. Photomixotrophic conditions, using containers with intermediate rate of gas exchange (yellow filter) and with 15–30?g L^?1 sucrose, produced an improvement in the growth traits and did not induce anatomical and physiological disturbances.     

Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?

Exposing plants to low VPD reduces leaf capacity to maintain adequate water status thereafter. To find the impact of VPD on functioning of stomata, stomatal morphology and leaf anatomy, fava bean plants were grown at low (L, 0.23 kPa) or moderate (M, 1.17 kPa) VPDs and some plants that developed their leaves at moderate VPD were then transferred for 4 days to low VPD (M→L). Part of the M→L‐plants were sprayed with ABA (abscisic acid) during exposure to L. L‐plants showed bigger stomata, larger pore area, thinner leaves and less spongy cells compared with M‐plants. Stomatal morphology (except aperture) and leaf anatomy of the M→L‐plants were almost similar to the M‐plants, while their transpiration rate and stomatal conductance were identical to that of L‐plants. The stomatal response to ABA was lost in L‐plants, but also after 1‐day exposure of M‐plants to low VPD. The level of foliar ABA sharply decreased within 1‐day exposure to L, while the level of ABA‐GE (ABA‐glucose ester) was not affected. Spraying ABA during the exposure to L prevented loss of stomatal closing response thereafter. The effect of low VPD was largely depending on exposure time: the stomatal responsiveness to ABA was lost after 1‐day exposure to low VPD, while the responsiveness to desiccation was gradually lost during 4‐day exposure to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main cause of loss of stomatal closure response to closing stimuli.     

Evaluation of the WHO classification of dengue disease severity during an epidemic in 2011 in the state of Ceará, Brazil

In 2009, the World Health Organization (WHO) issued a new guideline that stratifies dengue-affected patients into severe (SD) and non-severe dengue (NSD) (with or without warning signs). To evaluate the new recommendations, we completed a retrospective cross-sectional study of the dengue haemorrhagic fever (DHF) cases reported during an outbreak in 2011 in northeastern Brazil. We investigated 84 suspected DHF patients, including 45 (53.6%) males and 39 (46.4%) females. The ages of the patients ranged from five-83 years and the median age was 29. According to the DHF/dengue shock syndrome classification, 53 (63.1%) patients were classified as having dengue fever and 31 (36.9%) as having DHF. According to the 2009 WHO classification, 32 (38.1%) patients were grouped as having NSD [4 (4.8%) without warning signs and 28 (33.3%) with warning signs] and 52 (61.9%) as having SD. A better performance of the revised classification in the detection of severe clinical manifestations allows for an improved detection of patients with SD and may reduce deaths. The revised classification will not only facilitate effective screening and patient management, but will also enable the collection of standardised surveillance data for future epidemiological and clinical studies.     

Renal Function in Hepatosplenic Schistosomiasis – An Assessment of Renal Tubular Disorders

Background

Renal involvement in Schistosoma mansoni infection is not well studied. The aim of this study is to investigate the occurrence of renal abnormalities in patients with hepatosplenic schistosomiasis (HSS), especially renal tubular disorders.

Methods

This is a cross-sectional study with 20 consecutive patients with HSS followed in a medical center in Maceió, Alagoas, Brazil. Urinary acidification and concentration tests were performed using calcium chloride (CaCl₂) after a 12-h period of water and food deprivation. The biomarker monocyte chemoattractant protein 1 (MCP-1) was quantified in urine. Fractional excretion of sodium (FE_Na+), transtubular potassium gradient (TTKG) and solute-free water reabsorption (TcH₂O) were calculated. The HSS group was compared to a group of 17 healthy volunteers.

Results

Patients'' mean age and gender were similar to controls. Urinary acidification deficit was found in 45% of HSS patients. Urinary osmolality was significantly lower in HSS patients (588±112 vs. 764±165 mOsm/kg, p = 0,001) after a 12-h period of water deprivation. TcH₂O was lower in HSS patients (0.72±0.5 vs. 1.1±0.3, p = 0.04). Urinary concentration deficit was found in 85% of HSS patients. The values of MCP-1 were higher in HSS group than in control group (122±134 vs. 40±28 pg/mg-Cr, p = 0.01) and positively correlated with the values of microalbuminuria and proteinuria.

Conclusions

HSS is associated with important kidney dysfunction. The main abnormalities found were urinary concentrating ability and incomplete distal acidification defect, demonstrating the occurrence of tubular dysfunction. There was also an increase in urinary MCP-1, which appears to be a more sensitive marker of renal damage than urinary albumin excretion rate.     

High levels of Cellular Prion Protein improve astrocyte development

Prion protein (PrP^C) has neuroprotective functions and herein we demonstrate that astrocytes from PrP^C-over-expressing mice are more resistant to induced cell death than wild-type astrocytes. The Stress-Inducible-Protein 1 (STI1), a PrP^C ligand, prevents cell death in both wild-type and PrP^C-over-expressing astrocytes through the activation of protein-kinase-A. Cultured embryonic astrocytes and brain extracts from PrP^C-over-expressing mice show higher glial fibrillary acidic protein expression and reduced vimentin and nestin levels when compared to wild-type astrocytes, suggesting faster astrocyte maturation in the former mice. Our data indicate that PrP^C levels modulate astrocyte development, and that PrP^C–STI1 interaction contributes to protect against astrocyte death.     

Update on the challenging role of biofilms in peritoneal dialysis

Biofilms are commonly associated with an increased risk of patient infection. In peritoneal dialysis (PD), catheter associated infection, especially peritonitis, remains a clinically relevant problem. Although the presence of a biofilm is recognized in relapsing, repeat, and catheter-related peritonitis, it remains poorly characterized. In this review, an update on the role of biofilms in PD infections is presented. The emerging concept that host cells and tissue associated biofilms, in addition to the biofilms on the catheters themselves, contribute to the recalcitrance of infections is discussed. Furthermore, the evidence of biofilms on PD catheters, their developmental stages, and the possible influence of the PD environment are reviewed. The focus is given to ex vivo and in vitro studies that contribute to the elucidation of the interplay between host, microbial, and dialysis factors. The key issues that are still to be answered and the challenges to clinical practice are discussed.     

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.