Maria Maximovna Lukina

BACKGROUND: The E3 ubiquitin ligase TRIM29 is involved in basal epithelial development, cellular response to viral infection, and DNA damage. Furthermore, this protein can have both oncogenic and tumor suppressor properties. However, the molecular mechanisms of TRIM29 involvement in such a wide range of biological processes remain unclear. AIM: To identify protein partners of TRIM29 and its truncated forms and to determine the key molecular processes in which it is involved. MATERIALS AND METHODS: Cell cultures of normal prostate basal epithelium with overexpression of a full length TRIM29-FLAG chimeric protein or its truncated forms without the B-box or Coiled-Coil domain were obtained. Subsequently, protein partners of TRIM29 and truncated forms of TRIM29 were identified by protein immunoprecipitation followed by proteomic analysis (high-performance liquid chromatography with tandem mass spectrometry). Results were confirmed by Western blot and immunocytochemistry. RESULTS: TRIM29 binds to 288 proteins in the normal basal epithelium of the prostate. Deletion of the B-box has little effect on TRIM29 protein-protein interactions, whereas deletion of the Coiled-Coil domain deprives TRIM29 of most of its protein partners and impairs its dimerization. TRIM29 was found to localize to both the nucleus and cytoplasm, while deletion of functional domains does not prevent localization to different compartments, but does affect binding to proteins specific to these compartments. TRIM29 binds to cytoskeleton proteins, cellular stress response proteins, and RNA-binding proteins. In addition, TRIM29 is shown to increase cell resistance to genotoxic agents and to affect RNA splicing. CONCLUSION: Proteomic analysis showed that in normal prostate basal epithelium, E3 ubiquitin ligase TRIM29 binds to a relatively large number of proteins that perform different functions in different cell compartments. Our results are consistent with results obtained by other research teams who showed that TRIM29 is actively involved in cytoskeletal remodeling, cellular response to viral infection, and DNA damage. In addition, it was shown for the first time that TRIM29 interacts with stress granule proteins and RNA binding proteins and is able to regulate RNA splicing, and the Coiled-Coil domain of TRIM29 may play a key role in this process.

High-grade serous ovarian carcinoma (HGSOC) remains the most common and deadly form of ovarian cancer. However, available cell lines usually fail to appropriately represent its complex molecular and histological features. To overcome this drawback, we established OVAR79, a new cell line derived from the ascitic fluid of a patient with a diagnosis of HGSOC, which adds a unique set of properties to the study of ovarian cancer. In contrast to the common models, OVAR79 expresses TP53 without the common hotspot mutations and harbors the rare combination of mutations in both PIK3CA and PTEN genes, together with high-grade chromosomal instability with multiple gains and losses. These features, together with the high proliferation rate, ease of cultivation, and exceptional transfection efficiency of OVAR79, make it a readily available and versatile tool for various studies in the laboratory. We extensively characterized its growth, migration, and sensitivity to platinum- and taxane-based treatments in comparison with the commonly used SKOV3 and OVCAR3 ovarian cell lines. In summary, OVAR79 is an excellent addition for basic and translational ovarian cancer research and offers new insights into the biology of HGSOC.

Despite the growing interest in pre-mRNA alternative splicing (AS) as a therapeutic anticancer target, the potential of splicing inhibitors in treating solid tumors remains largely unexplored. We conducted a meta-analysis of transcriptome data from six different tumor types and revealed that splicing inhibitors induced similar patterns of AS, resulting in widespread exon-skipping and intron retention events that often lead to nonsense-mediated decay of the transcripts. Interestingly, in many cases exon skipping is induced by a compensatory cellular response to splicing inhibitor treatment. It involves an upregulation of multiple splicing factors and incomplete recognition of branch points by U2 snRNP. These post transcriptional changes downregulate one-third of essential DNA repair genes, thereby creating a therapeutic vulnerability that can be exploited for cancer treatment. To harness this vulnerability, we proposed a new approach to cancer treatment consisting of sequential addition of a splicing inhibitors followed by a DNA-damaging agent. Our in vitro and in vivo experiments demonstrated that this strategy exhibits promising therapeutic potential for a wide range of tumors.

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.

New water-soluble polynorbornenes P1–P4 containing oligoether, amino acid groups and luminophoric complexes of iridium(III) were synthesized by ring-opening metathesis polymerization. The polymeric products in organic solvents and in water demonstrate intense photoluminescence in the red spectral region. The polymers P1 and P3 with 1-phenylisoquinoline cyclometalating ligands in iridium fragments reveal 4–6 fold higher emission quantum yields in solutions than those of P2 and P4 that contain iridium complexes with 1-(thien-2-yl)isoquinoline cyclometalating ligands. The emission parameters of P1–P4 in degassed solutions essentially differ from those in the aerated solutions showing oxygen-dependent quenching of phosphorescence. Biological testing of P1 and P3 demonstrates that the polymers do not penetrate into live cultured cancer cells and normal skin fibroblasts and do not possess cytotoxicity within the concentrations and time ranges reasonable for biological studies. In vivo, the polymers display longer phosphorescence lifetimes in mouse tumors than in muscle, as measured using phosphorescence lifetime imaging (PLIM), which correlates with tumor hypoxia. Therefore, preliminary evaluation of the synthesized polymers shows their suitability for noninvasive in vivo assessments of oxygen levels in biological tissues.

Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic chemistry. We have designed and synthetized four novel iridium [Ir(N^C)₂(N^N)]⁺ complexes (N^C–benzothienyl-phenanthridine based cyclometalated ligand; N^N–pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters’ solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry. The complexes exhibited excitation close to the biological “window of transparency”, NIR emission at 730 nm, and quantum yields up to 12% in water. The compounds with higher degree of the chromophore shielding possess low toxicity, bleaching stability, absence of sensitivity to variations of pH, serum, and complex concentrations. The properties of these probes as oxygen sensors for biological systems have been studied by using phosphorescence lifetime imaging experiments in different cell cultures. The results showed essential lifetime response onto variations in oxygen concentration (2.0–2.3 μs under normoxia and 2.8–3.0 μs under hypoxia conditions) in complete agreement with the calibration curves obtained “in cuvette”. The data obtained indicate that these emitters can be used as semi-quantitative oxygen sensors in biological systems.

SPIE Digital Library Proceedings

SPIE Digital Library Proceedings

Viscosity, a reciprocal of fluidity, plays an important role in the functioning of living cells, as it is one of the key parameters for the regulation of their morphological and physiological state. In particular, the viscosity of the cytoplasmic membrane is thought to be involved in controlling the transport of molecules, permeability, biosynthesis, and catalytic activity of membrane enzymes.¹

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.