Wnts need a p(assport)24 to leave the ER

Wnts are secreted through a dedicated exocytic pathway, which has been only partly characterized. Here, Palmer and colleagues comment on two recent reports by the groups of K. Basler and M. Boutros, respectively, in which they show that p24 proteins take part in this exocytic route and are required for Wnt exit from the ER to the Golgi.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.212Wnt signalling proteins regulate diverse cellular processes during development and homeostasis. Since misregulation of Wnt signalling is associated with cancer, most research has been aimed at characterizing the signal transduction pathway. Recently, attention has focused on Wnt production due to the identification of factors required specifically for Wnt secretion. For instance, the specific requirement of Evi, also known as Wntless or Sprinter, suggested that Wnts might follow a specialized secretory route (Banziger et al, 2006; Bartscherer et al, 2006). Two recent papers published in EMBO reports by the groups of Konrad Basler in last month''s issue, and Michael Boutros in this issue, show that Wnt secretion requires the activity of p24 family members (Buechling et al, 2011; Port et al, 2011). Therefore, the specialized route might begin at endoplasmic reticulum (ER) exit sites.Wnts are secreted glycoproteins that can act many cell diameters from their source of production. Most, but not all Wnt proteins, are acylated and thus associate tightly with cellular membranes. Despite this association, acylated Wnts can be released from secreting cells and spread in the extracellular space (Bartscherer & Boutros, 2008; Port & Basler, 2010). Acylation of Wnts, which occurs in the ER, is thought to be mediated by the N-acetyl transferase encoded by porcupine (porc; van den Heuvel et al, 1993). After acylation, Wnt proteins associate with Evi, a multipass transmembrane protein found mostly at the Golgi and the plasma membrane (Fig 1A). This association is essential for the secretion of the Wnts that are acylated since, in the absence of Evi, they accumulate on internal membranes (Banziger et al, 2006; Bartscherer et al, 2006). It is therefore thought that acylated Wnts require Evi to exit the Golgi and progress to the cell surface (Port et al, 2008). This does not seem to be a requirement for non-lipidated Wnts, such as Drosophila WntD, which are secreted in the absence of Evi (Ching et al, 2008). Similarly, secretion of other signalling proteins proceeds normally without Evi. After reaching the cell surface, Evi is likely to have a choice between various routes. One such route, which involves the retromer complex, takes it back to the Golgi where it can participate in another round of Wnt secretion (Fig 1A). Alternatively, Evi can be targeted to lysosomes (Bartscherer & Boutros, 2008; Port & Basler, 2010). The factors that determine Evi transport remain poorly understood. Nevertheless, these studies highlight the essential and specific role of Evi for the secretion of lipidated Wnts.Open in a separate windowFigure 1Model summarizing the suggested roles of p24 proteins in endoplasmic reticulum to Golgi transport of Wg. (A) Schematic of a cell showing the current model of the Wg secretory pathway. Wg is produced in the ER where it is lipid-modified by Porc, and moved to the Golgi with the assistance of p24 proteins. In the Golgi, Wg joins Evi, which facilitates Wg transport to the cell surface. Evi is then recycled back to the Golgi in a path mediated by the retromer complex. (B)(i) In the absence of p24 proteins, there is no recruitment of Wg to COPII-coated vesicles and therefore a block of secretion in the ER. (ii) Port et al (2011) propose a similar model in which CHOp24/Emp24 and Éclair are involved in Wg recruitment, although only CHOp24/Emp24 binds to Wg. (iii) According to Buechling et al (2011), Opm recruits Wg to COPII-coated vesicles for movement to the Golgi. CHOp24 and p24-1 are also required for this process. ER, endoplasmic reticulum.Two groups have now reported the use of cell-based RNA interference (RNAi) screens to identify further proteins required for the secretion of Wingless (Wg), the main Drosophila Wnt (Buechling et al, 2011; Port et al, 2011). They found that p24 family members, a group of proteins previously implicated in both retrograde and anterograde transport between the ER and Golgi (Strating & Martens, 2009), are required for Wg secretion by S2 cells. Similarly, knockdown by transgenic RNAi shows that p24 proteins are required for normal levels of Wg secretion in Drosophila wing imaginal discs (Buechling et al, 2011; Port et al, 2011). As with Evi, this requirement seems to be relatively specific, since general secretion and the secretion of other signalling proteins, including the lipid-modified morphogen Hedgehog, are unaffected by p24 knockdown. Buechling et al also assessed the role of p24 proteins in WntD secretion. They found that RNAi against opossum (opm), one of the p24 members, prevents WntD secretion in cultured cells. They also show that the phenotypes of opm mutants and WntD mutant embryos resemble each other (Buechling et al, 2011). Therefore, while Evi is specifically required for the secretion of acylated Wnts, p24 proteins could contribute to the secretion of all Wnts. This function is likely to be conserved since the mammalian homologue of Opm, TMED5, is required for Wnt1 signalling, at least in a mammalian cell culture assay (Buechling et al, 2011).To gain understanding of the role of p24 proteins in Wnt secretion, both groups analysed the subcellular localization of Wg following p24 knockdown. They found accumulation in the ER and concomitant depletion in the Golgi, as indicated by reduced co-localization with Golgi markers (Fig 1Bi). They also found that p24 knockdown prevents Wg from stabilizing Evi in producing cells, suggesting that the stabilizing influence of Wg requires its exit from the ER (Buechling et al, 2011; Port et al, 2011). These results lead the authors to propose that the loss of p24 prevents the transport of Wg from the ER to the Golgi. Importantly, immunoprecipitation experiments suggest that Wg might interact physically with Opm and Emp24 (also known as CHOp24). This led both sets of authors to postulate a model whereby p24 proteins act as cargo receptors to escort Wnt proteins from the ER to the Golgi, whereupon they can bind to Evi, which will escort them to the plasma membrane. Thus, in this context, p24 proteins seem to have an anterograde function.Although both studies highlight the role of p24 proteins in Wnt secretion, they disagree on the relative importance of the various family members. Among the nine predicted p24 proteins encoded by the Drosophila genome, only Éclair and Emp24/CHOp24 were found to be required for Wg secretion by Port et al (2011; Fig 1Bii). By contrast, Buechling et al found that Opm, Emp24/CHOp24 and p24-1 all play a role in Wg secretion (fig 1Biii). Thus, only Emp24/CHOp24 is found by both groups to be essential for Wg secretion. Although functional redundancy among p24 proteins could explain why the removal of a single p24 protein has a relatively weak phenotype, there is no simple explanation as to why the very similar assays used by the two groups do not lead to identical conclusions. These differences could be worked out by the exchange of reagents and protocols.Regardless of the discrepancies, the two studies provide an important step in our understanding of Wnt secretion by demonstrating that Wnts engage with specialized components of the secretory machinery as early as in the ER. It might be relevant that the anterograde function of p24 proteins is directed at glycophosphatidylinositol (GPI)-anchored proteins, which have been shown to partition in raft-like microdomains (Strating & Martens, 2009). It is conceivable that GPI-anchored proteins, as well as Wnts, gather in a subdomain of the ER where they could both interact with p24 proteins and set off along their specialized secretory pathways. Wnt targeting to specialized membrane domains could in principle be mediated by their lipid moieties (Bartscherer & Boutros, 2008; Port & Basler, 2010). However, the process might turn out to be more complex if it is confirmed that p24 proteins are also required for the secretion of non-acylated Wnts (for example, WntD), as suggested by Buechling et al. In any case, it will be interesting to determine the precise molecular mechanism underlying the functional interaction between Wnts and p24 proteins as it is likely to explain how Wnts are allowed to exit the ER and start their journey out of the cell.     

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.     

Steep differences in wingless signaling trigger Myc-independent competitive cell interactions

Wnt signaling is a key regulator of development that?is often associated with cancer. Wingless,?a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, we found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. We suggest that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition.     

Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumor

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-β, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.     

A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent

From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.     

Down-regulation of ECRG4, a candidate tumor suppressor gene, in human breast cancer

Introduction

ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer.

Methods

Using DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n = 1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival.

Results

ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR = 0.84 [0.76–0.92], p = 0.0002) and overall survival (OS; HR = 0.72 [0.63–0.83], p = 5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS.

Conclusions

Our data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach.     

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.     

Tuberculosis in HIV-negative and HIV-infected patients in a low-incidence country: clinical characteristics and treatment outcomes

Background

In Switzerland and other developed countries, the number of tuberculosis (TB) cases has been decreasing for decades, but HIV-infected patients and migrants remain risk groups. The aim of this study was to compare characteristics of TB in HIV-negative and HIV-infected patients diagnosed in Switzerland, and between coinfected patients enrolled and not enrolled in the national Swiss HIV Cohort Study (SHCS).

Methods and Findings

All patients diagnosed with culture-confirmed TB in the SHCS and a random sample of culture-confirmed cases reported to the national TB registry 2000–2008 were included. Outcomes were assessed in HIV-infected patients and considered successful in case of cure or treatment completion. Ninety-three SHCS patients and 288 patients selected randomly from 4221 registered patients were analyzed. The registry sample included 10 (3.5%) coinfected patients not enrolled in the SHCS: the estimated number of HIV-infected patients not enrolled in the SHCS but reported to the registry 2000–2008 was 146 (95% CI 122–173). Coinfected patients were more likely to be from sub-Saharan Africa (51.5% versus 15.8%, P<0.0001) and to present disseminated disease (23.9% vs. 3.4%, P<0.0001) than HIV-negative patients. Coinfected patients not enrolled in the SHCS were asylum seekers or migrant workers, with lower CD4 cell counts at TB diagnosis (median CD4 count 79 cells/µL compared to 149 cells/µL among SHCS patients, P = 0.07). There were 6 patients (60.0%) with successful outcomes compared to 82 (88.2%) patients in the SHCS (P = 0.023).

Conclusions

The clinical presentation of coinfected patients differed from HIV-negative TB patients. The number of HIV-infected patients diagnosed with TB outside the SHCS is similar to the number diagnosed within the cohort but outcomes are poorer in patients not followed up in the national cohort. Special efforts are required to address the needs of this vulnerable population.