Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics.

Structure and function of cancer-related developmentally regulated GTP-binding protein 1 (DRG1) is conserved between sponges and humans.

Cancer is a disease caused by errors within the multicellular system and it represents a major health issue in multicellular organisms. Although cancer research has advanced substantially, new approaches focusing on fundamental aspects of cancer origin and mechanisms of spreading are necessary. Comparative genomic studies have shown that most genes linked to human cancer emerged during the early evolution of Metazoa. Thus, basal animals without true tissues and organs, such as sponges (Porifera), might be an innovative model system for understanding the molecular mechanisms of proteins involved in cancer biology. One of these proteins is developmentally regulated GTP-binding protein 1 (DRG1), a GTPase stabilized by interaction with DRG family regulatory protein 1 (DFRP1). This study reveals a high evolutionary conservation of DRG1 gene/protein in metazoans. Our biochemical analysis and structural predictions show that both recombinant sponge and human DRG1 are predominantly monomers that form complexes with DFRP1 and bind non-specifically to RNA and DNA. We demonstrate the conservation of sponge and human DRG1 biological features, including intracellular localization and DRG1:DFRP1 binding, function of DRG1 in α-tubulin dynamics, and its role in cancer biology demonstrated by increased proliferation, migration and colonization in human cancer cells. These results suggest that the ancestor of all Metazoa already possessed DRG1 that is structurally and functionally similar to the human DRG1, even before the development of real tissues or tumors, indicating an important function of DRG1 in fundamental cellular pathways.

Genetics of Marine Organisms Associated with Human Health.

Marine habitats harbour a large variety of organisms that belong to diverse taxa; from bacteria and unicellular eukaryotes to fungi, animals, and plants. Although we have only started to understand the diversity and structure of marine communities, it is clear that numerous marine species have or might have an impact on human health. Some are a source of natural products with potential or actual medical applications, others are toxic and harmful to humans, and some are used in biomedical research to help understand the molecular basis of human diseases. New molecular genetics and genomic methods provide powerful and ever more indispensable tools for studying marine organisms and all aspects of their influence on human health. Herein, we present work using the latest research, which mostly uses genomics, to tackle the questions related with the topic of the issue.

Rho Family of Ras-Like GTPases in Early-Branching Animals.

Non-bilaterian animals consist of four phyla; Porifera, Cnidaria, Ctenophora, and Placozoa. These early-diverging animals are crucial for understanding the evolution of the entire animal lineage. The Rho family of proteins make up a major branch of the Ras superfamily of small GTPases, which function as key molecular switches that play important roles in converting and amplifying external signals into cellular responses. This review represents a compilation of the current knowledge on Rho-family GTPases in non-bilaterian animals, the available experimental data about their biochemical characteristics and functions, as well as original bioinformatics analysis, in order to gain a general insight into the evolutionary history of Rho-family GTPases in simple animals.

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage.

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Characterization of Nme5-Like Gene/Protein from the Red Alga Chondrus Crispus.

The Nme gene/protein family of nucleoside diphosphate kinases (NDPK) was originally named after its member Nm23-H1/Nme1, the first identified metastasis suppressor. Human Nme proteins are divided in two groups. They all possess nucleoside diphosphate kinase domain (NDK). Group I (Nme1-Nme4) display a single type NDK domain, whereas Group II (Nme5-Nme9) display a single or several different NDK domains, associated or not associated with extra-domains. Data strongly suggest that, unlike Group I, none of the members of Group II display measurable NDPK activity, although some of them autophosphorylate. The multimeric form is required for the NDPK activity. Group I proteins are known to multimerize, while there are no data on the multimerization of Group II proteins. The Group II ancestral type protein was shown to be conserved in several species from three eukaryotic supergroups. Here, we analysed the Nme protein from an early branching eukaryotic lineage, the red alga Chondrus crispus. We show that the ancestral type protein, unlike its human homologue, was fully functional multimeric NDPK with high affinity to various types of DNA and dispersed localization throughout the eukaryotic cell. Its overexpression inhibits both cell proliferation and the anchorage-independent growth of cells in soft agar but fails to deregulate cell apoptosis. We conclude that the ancestral gene has changed during eukaryotic evolution, possibly in correlation with the protein function.

Sponges: A Reservoir of Genes Implicated in Human Cancer.

Recently, it was shown that the majority of genes linked to human diseases, such as cancer genes, evolved in two major evolutionary transitions-the emergence of unicellular organisms and the transition to multicellularity. Therefore, it has been widely accepted that the majority of disease-related genes has already been present in species distantly related to humans. An original way of studying human diseases relies on analyzing genes and proteins that cause a certain disease using model organisms that belong to the evolutionary level at which these genes have emerged. This kind of approach is supported by the simplicity of the genome/proteome, body plan, and physiology of such model organisms. It has been established for quite some time that sponges are an ideal model system for such studies, having a vast variety of genes known to be engaged in sophisticated processes and signalling pathways associated with higher animals. Sponges are considered to be the simplest multicellular animals and have changed little during evolution. Therefore, they provide an insight into the metazoan ancestor genome/proteome features. This review compiles current knowledge of cancer-related genes/proteins in marine sponges.

Nme family of proteins--clues from simple animals.

Nucleoside-diphosphate kinases (Nme/Nm23/NDPK) are evolutionarily conserved enzymes involved in many biological processes in vertebrates. The biochemical mechanisms of these processes are still largely unknown. The Nme family consists of ten members in humans of which Nme1/2 have been extensively studied in the context of carcinogenesis, especially metastasis formation. Lately, it has been proven that the majority of genes linked to human diseases were already present in species distantly related to humans. Most of cancer-related protein domains appeared during the two main evolutionary transitions-the emergence of unicellular eukaryotes and the transition to multicellular metazoans. In spite of these recent insights, current knowledge about cancer and status of cancer-related genes in simple animals is limited. One possible way of studying human diseases relies on analyzing genes/proteins that cause a certain disease by using model organism that represent the evolutionary level at which these genes have emerged. Therefore, basal metazoans are ideal model organisms for gaining a clearer picture how characteristics and functions of Nme genes changed in the transition to multicellularity and increasing complexity in animals, giving us exciting new evidence of their possible functions in potential pathological conditions in humans.

Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.