Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

Background

Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.

Results

Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1^-/-; Tp53^-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.

Conclusions

We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0428-9) contains supplementary material, which is available to authorized users.     

Integrative analysis of high-throughput RNAi screen data identifies the FER and CRKL tyrosine kinases as new regulators of the mitogenic ERK-dependent pathways in transformed cells

Background

Cell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells.

Results

The functional siRNA screen of 714 kinases in HeLa cells identified 91 kinases implicated in the regulation of cell growth, most of them never being reported in previous whole-genome siRNA screens. Based on gene ontology annotations, we have further discriminated between two classes of kinases that, when suppressed, result in alterations of the mitotic index and provoke cell-cycle arrest. Extinguished kinases that lead to a low mitotic index mostly include kinases implicated in cytosolic signaling. In contrast, extinguished kinases that result in a high mitotic index mostly include kinases implicated in cell division. By mapping hit kinases in the PhosphPOINT phosphoprotein database, we generated scale-free networks consisting of 449 and 661 protein-protein interactions for kinases from low MI and high MI groups, respectively. Further analyses of the kinase interactomes revealed specific modules such as FER- and CRKL-containing modules that connect three members of the epidermal growth factor receptor (EGFR) family, suggesting a tight control of the mitogenic EGF-dependent pathway. Based on experimental studies, we confirm the involvement of these two kinases in the regulation of tumor cell growth.

Conclusion

Based on a combined approach of large kinome-wide siRNA screens and ontology annotations, our study identifies for the first time two kinase groups differentially implicated in the control of cell proliferation. We further demonstrate that integrative analysis of the kinase interactome provides key information which can be used to facilitate or optimize target design for new therapeutic strategies. The complete list of protein-protein interactions from the two functional kinase groups will provide a useful database for future investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1169) contains supplementary material, which is available to authorized users.     

Microtubule-associated protein 9 (Map9/Asap) is required for the early steps of zebrafish development

Microtubules are structural components of the cell cytoskeleton and key factors for mitosis and ciliogenesis in eukaryotes. The regulation of MT dynamics requires non-motor MAPs. We previously showed that, in human cells in culture, MAP9 (also named ASAP) is involved in MT dynamics and is essential for mitotic spindle formation and mitosis progression. Indeed, misexpression of MAP9 leads to severe mitotic defects and cell death. Here, we investigated the in vivo role of map9 during zebrafish development. Map9 is expressed mainly as a maternal gene. Within cells, Map9 is associated with the MT network of the mitotic spindle and with centrosomes. Morpholino-mediated depletion of map9 leads to early development arrest before completion of epiboly. Map9 localizes to the MT array of the YSL. This MT network is destroyed in Map9-depleted embryos, and injection of anti-map9 morpholinos directly in the nascent YSL leads to arrest of epiboly/gastrulation. Finally, map9 knockdown deregulates the expression of genes involved in endodermal differentiation, dorso–ventral and left–right patterning, and other MT-based functions. At low morpholino doses, the surviving embryos show dramatic developmental defects, spindle and mitotic defects, and increased apoptosis. Our findings suggest that map9 is a crucial factor in early zebrafish development by regulating different MT-based processes.     

Prevalence of the prothrombin G20210A polymorphism in the Lebanese population: use of a reverse hybridization strip assay approach

The factor II (prothrombin) G20210A gene polymorphism is the second most common SNP reported in VTE where it is associated with elevated plasma prothrombin levels and with a 3-fold increased risk. We studied the distribution of the G/G, G/A, and A/A genotypes of the Prothrombin G20210A gene mutation in the general Lebanese population using a novel technique in order to assess their prevalence, compare the results to previously reported data and to describe an available method that will permit easy and fast identification of the mutation. Prothrombin different genotypes were determined using the Cardiovascular Disease (CVD) StripAssay which is based on a Polymerase Chain Reaction-Reverse hybridization technique and DNA from 205 unrelated healthy donors from our HLA-bank was used. The prevalence of G/G, G/A, and A/A genotypes was found to be 98.54, 1.46, and 0%, respectively, with G and A allelic frequency of 99 and 1%, respectively. The sampled Lebanese population showed prothrombin genotypes distribution similar to Caucasians, and our results are comparable to other reports on the Lebanese healthy individuals. However, this is the first report on the prevalence of prothrombin G20210A mutation using this technique. Our results suggest that this approach is reliable and can be used as an assessment for thrombophilia profile. In addition, future investigations should be conducted to assess the contribution of the prothrombin G20210A mutation, on its own and in collaboration with other factors, in various clinical entities notably VTE.     

Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere

Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.In soilless cultures, the recycling of drainage water within a system is the consequence of new laws concerning water saving and limitation of pollution. Such closed systems minimize costs by conserving water and reducing fertilizer input; however, they may favor the dissemination of pathogens (13). When pathogens manage to enter recirculation systems, they are rapidly disseminated and may cause disease epidemics, particularly during periods of stress, e.g., stress due to high temperatures and/or to low levels of dissolved oxygen in the nutrient solution. Thus, numerous facultative pathogens commonly found in conventional cultures may become economically significant (53). Several of them, e.g., Pythium spp. and Phytophthora spp., are well adapted to the aquatic environment of hydroponic systems: they produce flagellate zoospores which enable them to swim in the nutrient solution, facilitating the spread of infection (18, 21, 36, 54, 61).Various methods are used to reduce the risks to plant health. Over recent years, the disinfection of nutrient solutions by physical or chemical treatments, e.g., ozonization, UV irradiation, chlorination, and thermo-disinfection, has been developed (13, 38). These methods effectively destroy pathogenic microorganisms but are harmful to species liable to benefit the plant, to be used as biocontrol agents, or both. Indeed, recirculation of nutrient solutions in closed hydroponic systems favors the establishment of a potentially suppressive microflora besides the pathogenic microflora (16, 28, 39, 41). The development of a beneficial microflora may thus be impeded by treatments used to destroy pathogenic microorganisms. Consequently, interest has been focused on the management of microorganisms in soilless cultures (12). Postma and coworkers (40) found that the extent of root disease is increased by the use of autoclaved rock wool. Tu and coworkers (59) observed that root rot disease was less severe in closed hydroponic systems than in open cultures and suggested that the difference was due to a higher density of bacteria in the closed systems. According to Paulitz (34), the diversity of microorganisms in soilless cultures is more limited than that in conventional soil cultures, such that conditions are more suitable for beneficial microorganisms, and consequently for effective biological control, in soilless than in conventional soil cultures.Biocontrol strategies are promising (7, 35). However, both biotic and abiotic factors may affect the performance of biocontrol methods. Relevant biotic factors include interactions with nontarget microorganisms (6), poor implantation of the biocontrol agent due to nonadaptation to the hydroponic system or resistance from the native microflora, shelf life and formulation, and host plant species and cultivar effects. Abiotic factors include climatic, chemical, and physical conditions of the soil or rhizosphere.Despite the limitations, various studies report evidence of the suppression of disease following the inoculation of hydroponic systems with antagonistic microorganisms. In particular, Pythium oligandrum is an effective biocontrol agent (2, 14, 49, 64). This oomycete colonizes roots without damaging the host plant cells (24, 45) and survives in the rhizosphere, where it exerts its biocontrol (57). P. oligandrum acts through both direct effects (mycoparasitism, antibiosis, and competition for nutrients and space) and indirect effects (stimulation of plant defense reactions and plant growth promotion) (49). The operating effects seem to depend on the type of pathogenic fungi being controlled (3, 48, 49). Le Floch and coworkers suggested that mycoparasitism is not the main mode of action (23). Root colonization by P. oligandrum may induce systemic resistance associated with the synthesis of elicitors protecting the plant from its aggressors (4, 17, 31, 37, 56). Several studies have investigated formulations of P. oligandrum oospores applied to soil or seeds, and their production and use, to optimize the efficacy of biocontrol (9, 30).Effective biocontrol by P. oligandrum may be limited by its heterogeneous implantation in the rhizosphere (46). Therefore, enhanced implantation and persistence of P. oligandrum in the rhizosphere should improve plant protection. We report an investigation of the persistence of P. oligandrum and its impact on the native fungal microflora of the roots. Three strains with characteristic traits were selected to constitute an inoculum applied to tomato plant roots. The characteristics of the strains were the production of oospores to allow root colonization and favor persistence, the synthesis of tryptamine, a plant growth enhancer (22), and the production of oligandrin, a plant-protective elicitor (37). The inoculated rhizospheres were monitored to evaluate the persistence of the strains and their effects on the microflora. The populations of the common tomato root pathogen P. dissotocum (endemic in the studied systems) and of P. oligandrum were both assessed by plate counting and real-time PCR. The strain(s) of P. oligandrum responsible for the colonization of the rhizosphere was identified by inter-simple-sequence-repeat (ISSR) methodology. Single-strand conformational polymorphism (SSCP) investigations were used to study the effects of P. oligandrum on the fungal populations colonizing the rhizosphere and the fungal dynamics throughout the cropping season.     

In Vivo Retargeting of Adenovirus Type 5 to αvβ6 Integrin Results in Reduced Hepatotoxicity and Improved Tumor Uptake following Systemic Delivery

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin αvβ6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, αvβ6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of αvβ6. The corresponding αvβ6-retargeted virus Ad5-EGFP_A20 exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of αvβ6-positive human carcinoma lines compared with Ad5-EGFP_WT. Using an αvβ6-positive (DX3-β6) xenograft model, we observed a ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP_A20 to improve delivery to αvβ6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.The aim of cancer gene therapy is to achieve targeted delivery of therapeutic transgenes to malignant tissue, with negligible effects on surrounding healthy tissue. Efforts in the development of adenoviruses as therapeutic agents have been persistent. However, several challenges still remain. Inefficient transduction of diseased tissue and the innate hepatotropism and toxicity of adenovirus type 5 (Ad5) in vivo following intravenous delivery represent major issues to be addressed. Additionally, the use of adenoviral vectors for cancer therapy is thought to be incompatible with the broad distribution of the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR), in normal tissues (6). Furthermore, it recently has emerged that human, but not murine, erythrocytes express CAR on their surface, which promotes the sequestration of Ad5 in the circulation and may represent another restriction to efficient tumor delivery in vivo (8).The predominant adenoviral serotype currently used in gene therapy applications is human Ad5. Ad5 binds to cells through a docking process in which the distal knob domain of the fiber structural protein binds to CAR (6, 23). This is followed by the exposure of an arginine-glycine-aspartate (RGD) motif in the penton base which promotes viral internalization, mediated primarily by αvβ3 and αvβ5 integrins (49). Binding to CAR represents the initial event in cell attachment in vitro, and therefore CAR expression levels long have been thought to be critical in determining the transduction efficiency of Ad5 in vivo. Several studies have reported low expression of CAR in primary carcinoma lines and tumor explants (3, 21, 30, 32, 37), highlighting the necessity for CAR-independent targeting strategies. However, the nonspecific sequestration of Ad5 in the liver remains the major obstacle to achieving high-efficiency tumor targeting following systemic delivery.A preeminent role for coagulation factors (i.e., FVII, FIX, FX, protein C, and C4BP) in directing liver uptake following systemic delivery has been demonstrated in recent years (36, 41, 48), and hepatocyte transduction now has been shown to be mediated predominantly by a direct Ad5 hexon-FX interaction (22, 48). This discovery has prompted the experimental use of anticoagulants, such as warfarin, in an attempt to avoid liver sequestration, with the aim of increasing the bioavailability of the virus for the tumor. However, it recently has emerged that coagulation factors may also be required for efficient tumor delivery in vivo and that the depletion of blood factors may in fact preclude successful tumor uptake (16). Accordingly, Ad vector constructs which combine liver detargeting with high-efficiency, CAR-independent gene delivery to cancer-specific receptors are highly desirable.The epithelial cell-specific integrin αvβ6 generally is undetectable in normal adult tissue but is upregulated significantly in numerous carcinomas, where high expression often correlates with poor prognosis (1, 4, 13). We have shown previously that over 90% of oral squamous cell carcinomas express αvβ6 strongly (35, 45) and that high αvβ6 expression promotes tumor progression (35, 43, 46). Binding to αvβ6 is via the RGD motif in its ligands, which include the latency-associated peptides (LAP) of transforming growth factor β1 (TGF-β1) and TGF-β3 (33), in addition to the VP1 structural protein of foot-and-mouth disease virus (FMDV), for which αvβ6 is a native receptor (20). We have shown that specificity for αvβ6 is dependent on the inclusion of a DLXXL motif in an extended carboxy α-helical loop, with the RGD motif situated at the apex of a hairpin loop domain (10). Functional analysis of known αvβ6 ligands identified a candidate peptide, A20FMDV2, which had high affinity and selective binding to αvβ6 (10). Recent studies have further supported this finding, demonstrating that this peptide forms a highly stable, EDTA-resistant complex with αvβ6 integrin, in a manner analogous to the highly infectious FMDV (11).Over the past decade, significant attempts to increase the delivery of Ad5 to target tissues have been made by modifying structural tropism determinants. Retargeting strategies have included the insertion of RGD motifs (12), polylysine (pK7) motifs (50) and TAT peptide from human immunodeficiency virus type 1 (26) in attempts to improve delivery to malignant tissue or to the endothelial networks that supply the tumors. The resolution of the crystal structure of the Ad5 knob domain by X-ray crystallography identified the HI loop region as being suitable for peptide incorporation (51), and to date this site has been shown to tolerate the insertion of ligands of up to 83 amino acids with negligible effects on structural integrity (5). In order to redirect the native tropism of Ad5 to αvβ6, we genetically incorporated A20FMDV2 into the HI loop region of the fiber knob domain. We hypothesized that Ad5-EGFP_A20 would permit significant improvements in selective delivery to αvβ6, both in vitro and in vivo.Here, we describe the successful subversion of Ad5 infection to αvβ6 expressed on human carcinoma cell lines in vitro and demonstrate that the enhanced transduction observed with Ad5-EGFP_A20 is CAR independent. We have confirmed that the improved infectivity is due to the insertion of the A20FMDV2 peptide and that cell entry is mediated predominantly through αvβ6 integrin. Additionally, using an αvβ6-positive (DX3-β6) xenograft model, we observed ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT in vivo following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. These data show that redirecting Ad5 to αvβ6 can increase tumor delivery while simultaneously limiting hepatotoxicity; this may therefore represent a means of overcoming some of the limitations of current Ad5 therapy.     

Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes

Background  

Functional screens based on dsRNA-mediated gene silencing identified several Anopheles gambiae genes that limit Plasmodium berghei infection. However, some of the genes identified in these screens have no effect on the human malaria parasite Plasmodium falciparum; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different Plasmodium species.