Biological evidence of cancer stem-like cells and recurrent disease in osteosarcoma

Sarcomas are a large family of cancers originating in the mesenchyme. Composed of more than 100 histological subtypes, soft tissue and bone sarcomas remain clinically challenging, particularly in children and adolescents in whom sarcomas are the second most common malignant entities. Osteosarcoma is the main primary bone tumor in adolescents and young adults and is characterized by a high propensity to induce distant metastatic foci and become multi-drug resistant. The innate and acquired resistance of osteosarcoma can be explained by high histological heterogeneity and genetic/molecular diversity. In the last decade, the notion of cancer stem-like cells (CSCs) has emerged. This subset of cancer cells has been linked to drug resistance properties, recurrence of the disease, and therapeutic failure. Although CSCs remain controversial, many elements are in favor of them playing a role in the development of the drug resistance profile. The present review gives a brief overview of the most recent biological evidence of the presence of CSCs in osteosarcomas and their role in the drug resistance profile of these rare oncological entities. Their use as promising therapeutic targets is discussed

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements. Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D. Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p < 0.05). Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins

Three-dimensional in vitro culture models in oncology research

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria

Virulence and Pathogen Multiplication: A Serial Passage Experiment in the Hypervirulent Bacterial Insect-Pathogen Xenorhabdus nematophila

The trade-off hypothesis proposes that the evolution of pathogens' virulence is shaped by a link between virulence and contagiousness. This link is often assumed to come from the fact that pathogens are contagious only if they can reach high parasitic load in the infected host. In this paper we present an experimental test of the hypothesis that selection on fast replication can affect virulence. In a serial passage experiment, we selected 80 lines of the bacterial insect-pathogen Xenorhabdus nematophila to multiply fast in an artificial culture medium. This selection resulted in shortened lag phase in our selected bacteria. We then injected these bacteria into insects and observed an increase in virulence. This could be taken as a sign that virulence in Xenorhabdus is linked to fast multiplication. But we found, among the selected lineages, either no link or a positive correlation between lag duration and virulence: the most virulent bacteria were the last to start multiplying. We then surveyed phenotypes that are under the control of the flhDC super regulon, which has been shown to be involved in Xenorhabdus virulence. We found that, in one treatment, the flhDC regulon has evolved rapidly, but that the changes we observed were not connected to virulence. All together, these results indicate that virulence is, in Xenorhabdus as in many other pathogens, a multifactorial trait. Being able to grow fast is one way to be virulent. But other ways exist which renders the evolution of virulence hard to predict

NEDDylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies

OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR

Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro