Interaction of the middle domains stabilizes Hsp90α dimer in a closed conformation with high affinity for p23.

The human genome encodes two highly similar cytosolic Hsp90 proteins called isoforms Hsp90α and Hsp90ß. Of the 300 client proteins for Hsp90 identified so far only a handful interact specifically with one Hsp90 isoform. Here we report for the first time that Hsp90 cochaperone p23 binds preferentially to Hsp90α and that this interaction is mediated by the middle domain of Hsp90α. Based on the homology modeling, we infer that the middle domains in the Hsp90α dimer bind stronger with each other than in the Hsp90ß dimer. Therefore, compared to Hsp90ß, Hsp90α may adopt closed conformation more easily. Hsp90 interacts with p23 in the closed conformation. Hsp90α binds human recombinant p23 about three times stronger than Hsp90ß but with significantly smaller exothermic enthalpy as determined by isothermal titration calorimetry of direct binding between the purified proteins. As p23 binds to Hsp90 in a closed conformation, stabilization of the Hsp90α dimer in the closed conformation by its middle domains explains preference of p23 to this Hsp90 isoform.

Middle domain of human Hsp90 isoforms differentially binds Aha1 in human cells and alters Hsp90 activity in yeast.

Hsp90 is an essential chaperone for more than 200 client proteins in eukaryotic cells. The human genome encodes two highly similar cytosolic Hsp90 proteins called Hsp90α and Hsp90ß. Most of the client proteins can interact with either Hsp90 protein; however, only a handful client proteins and one co-chaperone that interact specifically with one of the Hsp90 isoforms were identified. Structural differences underlying these isoform-specific interactions were not studied. Here we report for the first time that the Hsp90 co-chaperone Aha1 interacts preferentially with Hsp90α. The distinction depends on the middle domain of Hsp90. The middle domain of Hsp90α is also responsible for the slow growth phenotype of yeasts that express this isoform as a sole source of Hsp90. These results suggest that differences in the middle domain of Hsp90α and Hsp90ß may be responsible for the isoform-specific interactions with selected proteins. Also shown here within, we determine that preferential chaperoning of cIAP1 by Hsp90ß is mediated by the N-terminal domain of this isoform.

Cytidine does not affect acute toxicity of intravenously administered choline.

Cytidine-5'-diphosphocholine (CDP-choline) is a key precursor for the intracellular synthesis of phosphatidylcholine and other phospholipids. Following either intravenous or oral application citicoline (CDP-choline of exogenous origin) undergoes quick decomposition to cytidine and choline, and for this reason it is frequently considered a prodrug. However, upon acute intravenous application in mice citicoline is, on a molar basis, 20 times less toxic than choline. To find out whether cytidine may attenuate toxicity of choline, in the present experiments we compared maximum tolerated doses of single intravenous injections of choline and equimolar mixture of choline and cytidine. We assumed that, if after oral intake a substantial part of citicoline is catabolised already in the intestine and its catabolites enter blood separately, intravenously applied equimolar mixture of cytidine and choline will be markedly less toxic than an equivalent molar dose of choline. However, the maximum tolerated single doses determined in our experiment for choline and equimolar mixture of choline and cytidine were similar. These data suggest that citicoline taken orally is not significantly decomposed in the intestinal lumen, but absorbed to blood as the intact molecule.

From Molecular Biology to Novel Immunotherapies and Nanomedicine in Uveal Melanoma.

Molecular biology studies of uveal melanoma have resulted in the development of novel immunotherapy approaches including tebentafusp-a T cell-redirecting bispecific fusion protein. More biomarkers are currently being studied. As a result, combined immunotherapy is being developed as well as immunotherapy with bifunctional checkpoint inhibitory T cell engagers and natural killer cells. Current trials cover tumor-infiltrating lymphocytes (TIL), vaccination with IKKb-matured dendritic cells, or autologous dendritic cells loaded with autologous tumor RNA. Another potential approach to treat UM could be based on T cell receptor engineering rather than antibody modification. Immune-mobilizing monoclonal T cell receptors (TCR) against cancer, called ImmTAC TM molecules, represent such an approach. Moreover, nanomedicine, especially miRNA approaches, are promising for future trials. Finally, theranostic radiopharmaceuticals enabling diagnosis and therapy with the same molecule bring hope to this research.

Imaging of Uveal Melanoma-Current Standard and Methods in Development.

Uveal melanoma is the most common primary intraocular malignancy in adults, characterized by an insidious onset and poor prognosis strongly associated with tumor size and the presence of distant metastases, most commonly in the liver. Contrary to most tumor identification, a biopsy followed by a pathological exam is used only in certain cases. Therefore, an early and noninvasive diagnosis is essential to enhance patients' chances for early treatment. We reviewed imaging modalities currently used in the diagnostics of uveal melanoma, including fundus imaging, ultrasonography (US), optical coherence tomography (OCT), single-photon emission computed tomography (SPECT), fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), fundus autofluorescence (FAF), as well as positron emission tomography/computed tomography (PET/CT) or magnetic resonance imaging (MRI). The principle of imaging techniques is briefly explained, along with their role in the diagnostic process and a summary of their advantages and limitations. Further, the experimental data and the advancements in imaging modalities are explained. We describe UM imaging innovations, show their current usage and development, and explain the possibilities of utilizing such modalities to diagnose uveal melanoma in the future.

TP53 in Biology and Treatment of Osteosarcoma.

The TP53 gene is mutated in 50% of human tumors. Oncogenic functions of mutant TP53 maintain tumor cell proliferation and tumor growth also in osteosarcomas. We collected data on TP53 mutations in patients to indicate which are more common and describe their role in in vitro and animal models. We also describe animal models with TP53 dysfunction, which provide a good platform for testing the potential therapeutic approaches. Finally, we have indicated a whole range of pharmacological compounds that modulate the action of p53, stabilize its mutated versions or lead to its degradation, cause silencing or, on the contrary, induce the expression of its functional version in genetic therapy. Although many of the described therapies are at the preclinical testing stage, they offer hope for a change in the approach to osteosarcoma treatment based on TP53 targeting in the future.

New Perspectives for Eye-Sparing Treatment Strategies in Primary Uveal Melanoma.

Uveal melanoma is the most common intraocular malignancy and arises from melanocytes in the choroid, ciliary body, or iris. The current eye-sparing treatment options include surgical treatment, plaque brachytherapy, proton beam radiotherapy, stereotactic photon radiotherapy, or photodynamic therapy. However, the efficacy of these methods is still unsatisfactory. This article reviews several possible new treatment options and their potential advantages in treating localized uveal melanoma. These methods may be based on the physical destruction of the cancerous cells by applying ultrasounds. Two examples of such an approach are High-Intensity Focused Ultrasound (HIFU)-a promising technology of thermal destruction of solid tumors located deep under the skin and sonodynamic therapy (SDT) that induces reactive oxygen species. Another approach may be based on improving the penetration of anti-cancer agents into UM cells. The most promising technologies from this group are based on enhancing drug delivery by applying electric current. One such approach is called transcorneal iontophoresis and has already been shown to increase the local concentration of several different therapeutics. Another technique, electrically enhanced chemotherapy, may promote drug delivery from the intercellular space to cells. Finally, new advanced nanoparticles are developed to combine diagnostic imaging and therapy (i.e., theranostics). However, these methods are mostly at an early stage of development. More advanced and targeted preclinical studies and clinical trials would be needed to introduce some of these techniques to routine clinical practice.

Molecular Biology of Osteosarcoma.

Osteosarcoma (OS) is the most frequent primary bone cancer in children and adolescents and the third most frequent in adults. Many inherited germline mutations are responsible for syndromes that predispose to osteosarcomas including Li Fraumeni syndrome, retinoblastoma syndrome, Werner syndrome, Bloom syndrome or Diamond-Blackfan anemia. TP53 is the most frequently altered gene in osteosarcoma. Among other genes mutated in more than 10% of OS cases, c-Myc plays a role in OS development and promotes cell invasion by activating MEK-ERK pathways. Several genomic studies showed frequent alterations in the RB gene in pediatric OS patients. Osteosarcoma driver mutations have been reported in NOTCH1, FOS, NF2, WIF1, BRCA2, APC, PTCH1 and PRKAR1A genes. Some miRNAs such as miR-21, -34a, -143, -148a, -195a, -199a-3p and -382 regulate the pathogenic activity of MAPK and PI3K/Akt-signaling pathways in osteosarcoma. CD133+ osteosarcoma cells have been shown to exhibit stem-like gene expression and can be tumor-initiating cells and play a role in metastasis and development of drug resistance. Although currently osteosarcoma treatment is based on adriamycin chemoregimens and surgery, there are several potential targeted therapies in development. First of all, activity and safety of cabozantinib in osteosarcoma were studied, as well as sorafenib and pazopanib. Finally, novel bifunctional molecules, of potential imaging and osteosarcoma targeting applications may be used in the future.

TP53-Deficient Angiosarcoma Expression Profiling in Rat Model.

Sarcomas are a heterogeneous group of malignant tumors, that develop from mesenchymal cells. Sarcomas are tumors associated with poor prognosis and expected short overall survival. Efforts to improve treatment efficacy and treatment outcomes of advanced and metastatic sarcoma patients have not led to significant improvements in the last decades. In the Tp53C273X/C273X rat model we therefore aimed to characterize specific gene expression pattern of angiosarcomas with a loss of TP53 function. The presence of metabolically active tumors in several locations including the brain, head and neck, extremities and abdomen was confirmed by magnetic resonance imaging (MRI) and positron emission tomography (PET) examinations. Limb angiosarcoma tumors were selected for microarray expression analysis. The most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis, chromosome, nucleosome and telomere maintenance as well as DNA replication and recombination. The downregulated genes were responsible for metabolism, including respiratory chain electron transport, tricarboxylic acid (TCA) cycle, fatty acid metabolism and amino-acid catabolism. Our findings demonstrated that the type of developing sarcoma depends on genetic background, underscoring the importance of developing more malignancy susceptibility models in various strains and species to simulate the study of the diverse genetics of human sarcomas.

Choosing The Right Animal Model for Renal Cancer Research.

The increase in the life expectancy of patients with renal cell carcinoma (RCC) in the last decade is due to changes that have occurred in the area of preclinical studies. Understanding cancer pathophysiology and the emergence of new therapeutic options, including immunotherapy, would not be possible without proper research. Before new approaches to disease treatment are developed and introduced into clinical practice they must be preceded by preclinical tests, in which animal studies play a significant role. This review describes the progress in animal model development in kidney cancer research starting from the oldest syngeneic or chemically-induced models, through genetically modified mice, finally to xenograft, especially patient-derived, avatar and humanized mouse models. As there are a number of subtypes of RCC, our aim is to help to choose the right animal model for a particular kidney cancer subtype. The data on genetic backgrounds, biochemical parameters, histology, different stages of carcinogenesis and metastasis in various animal models of RCC as well as their translational relevance are summarized. Moreover, we shed some light on imaging methods, which can help define tumor microstructure, assist in the analysis of its metabolic changes and track metastasis development.

Citicoline: A Superior Form of Choline?

Medicines containing citicoline (cytidine-diphosphocholine) as an active principle have been marketed since the 1970s as nootropic and psychostimulant drugs available on prescription. Recently, the inner salt variant of this substance was pronounced a food ingredient in the major world markets. However, in the EU no nutrition or health claim has been authorized for use in commercial communications concerning its properties. Citicoline is considered a dietetic source of choline and cytidine. Cytidine does not have any health claim authorized either, but there are claims authorized for choline, concerning its contribution to normal lipid metabolism, maintenance of normal liver function, and normal homocysteine metabolism. The applicability of these claims to citicoline is discussed, leading to the conclusion that the issue is not a trivial one. Intriguing data, showing that on a molar mass basis citicoline is significantly less toxic than choline, are also analyzed. It is hypothesized that, compared to choline moiety in other dietary sources such as phosphatidylcholine, choline in citicoline is less prone to conversion to trimethylamine (TMA) and its putative atherogenic N-oxide (TMAO). Epidemiological studies have suggested that choline supplementation may improve cognitive performance, and for this application citicoline may be safer and more efficacious.