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Background: MicroRNAs (miRNAs) are key regulators of gene expression that have been implicated in gynecological and breast cancers. Understanding the cancer stage-wise expression patterns of miRNAs and their interactions with other RNA molecules in cancer is crucial to improve cancer diagnosis and treatment planning. Comprehensive web tools that integrate data on the transcriptome, circulating miRNAs, and their validated targets to derive beneficial conclusions in cancer research are lacking. Methods: Using the Shiny R package, we developed a web tool called ExplORRNet that integrates transcriptomic profiles from The Cancer Genome Atlas and miRNA expression data derived from various sources, including tissues, cell lines, exosomes, serum, and plasma, available in the Gene Expression Omnibus database. Differential expression analyses between normal and tumor tissue samples as well as different stages of cancer, accompanied by gene enrichment and survival analyses, can be performed using specialized R packages. Additionally, a miRNA-messenger RNA (mRNA)-long non-coding RNA (lncRNA) networks are constructed to identify regulatory modules. Results: Our tool identifies cancer stage-wise differentially regulated miRNAs, mRNAs, and lncRNAs in gynecological and breast cancers. Survival analysis identifies miRNAs associated with patient survival, and functional enrichment analysis provides insights into dysregulated miRNA-related biological processes and pathways. The miRNA–mRNA–lncRNA networks highlight interconnected regulatory molecular modules driving cancer progression. Case studies demonstrate the utility of the ExplORRNet for studying gynecological and breast cancers. Conclusion: ExplORRNet is an intuitive and user-friendly web tool that provides a deeper understanding of dysregulated miRNAs and their functional implications in gynecological and breast cancers. We hope our ExplORRNet tool has potential utility among the clinical and basic researchers and will be beneficial to the entire cancer genomics community to encourage and facilitate mining the rapidly growing public databases to progress the field of precision oncology. The ExplORRNet is available at https://mirna.cs.ut.ee.

Abstract The choice of targeted therapies for treatment of glioblastoma patients is currently limited, and most glioblastoma patients die from the disease recurrence. Thus, systematic studies in simplified model systems are required to pinpoint the choice of targets for further exploration in clinical settings. Here, we report screening of 5 compounds targeting epigenetic writers or erasers and 6 compounds targeting cell cycle-regulating protein kinases against 3 glioblastoma cell lines following incubation under normoxic or hypoxic conditions. The viability/proliferation assay indicated that PRMT5 inhibitor onametostat was endowed with high potency under both normoxic and hypoxic conditions in cell lines that are strongly MGMT-positive (T98-G), weakly MGMT-positive (U-251 MG), or MGMT-negative (U-87 MG). In U-251 MG and U-87 MG cells, onametostat also affected the spheroid formation at concentrations lower than the currently used chemotherapeutic drug lomustine. In T98-G cell line, treatment with onametostat led to dramatic changes in the transcriptome profile by inducing the cell cycle arrest, suppressing RNA splicing, and down-regulating several major glioblastoma cell survival pathways. Further validation by immunostaining in three cell lines confirmed that onametostat affects cell cycle and causes reduction in nucleolar protein levels. In this way, inhibition of epigenetic targets might represent a viable strategy for glioblastoma treatment even in the case of decreased chemo- and radiation sensitivity, although further studies in clinically more relevant models are required.

Abstract Despite the use of multimodal treatment combinations, the prognosis of glioblastoma (GB) is still poor. To prevent rapid tumor recurrence, targeted strategies for the treatment of GB are widely sought. Here, we compared the efficacy of focused modulation of a set of signaling pathways in two GB cell lines, U-251 MG and T98-G, using a panel of thirteen compounds targeting cell cycle progression, proliferation, epigenetic modifications, and DNA repair mechanism. In parallel, we tested combinations of these compounds with temozolomide and lomustine, the standard chemotherapy agents used in GB treatment. Two major trends were found: within individual compounds, the lowest IC50 values were exhibited by the Aurora kinase inhibitors, whereas in the case of mixtures, the addition of DNA methyltransferase 1 inhibitor azacytidine to lomustine proved the most beneficial. The efficacy of cell cycle-targeting compounds was further augmented by combination with radiation therapy using two different treatment regimes. The potency of azacytidine and lomustine mixtures was validated using a unique assay pipeline that utilizes automated imaging and machine learning-based data analysis algorithm for assessment of cell number and DNA damage extent. Based on our results, the combination of azacytidine and lomustine should be tested in GB clinical trials.

Abstract Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSCCtrl); however, this has not been assessed in PCOS cells (eSCPCOS). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs. non-DE eSCPCOS without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling. Overall, the results highlight an underlying defect in decidualized eSCPCOS, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.

Multiple studies have shown associations between exposure to endocrine disrupting chemicals (EDCs) and reduced fertility in women. However, little is known about the target organs of chemical disruption of female fertility. Here, we focus on the hormone-sensitive uterine lining, the endometrium, as a potential target. Decidualization is the morphological and functional change that endometrial stromal cells undergo to support endometrial receptivity, which is crucial for successful implantation, placentation, and pregnancy. We investigated the effect of nine selected EDCs on primary human endometrial stromal cell decidualization in vitro. The cells were exposed to a decidualization-inducing mixture in the presence or absence of 1 μM of nine different EDCs for nine days. Extent of decidualization was assessed by measuring the activity of cAMP dependent protein kinase, Rho-associated coiled-coil containing protein kinase, and protein kinase B in lysates using photoluminescent probes, and secretion of prolactin into the media by using ELISA. Decidualization-inducing mixture upregulated activity of protein kinases and prolactin secretion in cells derived from all women. Of the tested chemicals, dichlorodiphenyldichloroethylene (p,p’-DDE), hexachlorobenzene (HCB) and perfluorooctanesulfonic acid (PFOS) significantly reduced decidualization as judged by the kinase markers and prolactin secretion. In addition, bisphenol A (BPA) reduced prolactin secretion but did not significantly affect activity of the kinases. None of the EDCs was cytotoxic, based on the assessment of total protein content or activity of the viability marker casein kinase 2 in lysates. These results indicate that EDCs commonly present in the blood circulation of reproductive-aged women can reduce decidualization of human endometrial stromal cells in vitro. Future studies should focus on detailed hazard assessment to define possible risks of EDC exposure to endometrial dysfunction and implantation failure in women.

Immune checkpoint inhibitors are increasingly used in combination with chemotherapy for the treatment of non-small cell lung cancer, yet the success of combination therapies is relatively limited. Thus, more detailed insight regarding the tumor molecular markers that may affect the responsiveness of patients to therapy is required. Here, we set out to explore the proteome of two lung adenocarcinoma cell lines (HCC-44 and A549) treated with cisplatin, pemetrexed, durvalumab, and the corresponding mixtures to establish the differences in post-treatment protein expression that can serve as markers of chemosensitivity or resistance. The mass spectrometry study showed that the addition of durvalumab to the treatment mixture resulted in cell line- and chemotherapeutic agent-dependent responses and confirmed the previously reported involvement of DNA repair machinery in the potentiation of the chemotherapy effect. Further validation using immunofluorescence also indicated that the potentiating effect of durvalumab in the case of cisplatin treatment was dependent on the tumor suppressor RB-1 in the PD-L1 weakly positive cells. In addition, we identified aldehyde dehydrogenase ALDH1A3 as the general putative resistance marker. Further studies in patient biopsy samples will be required to confirm the clinical significance of these findings.

Bivalent ligands, including bisubstrate inhibitors, are conjugates of pharmacophores, which simultaneously target two binding sites of the biomolecule. Such structures offer attainable means for the development of compounds whose ability to bind to the biological target could be modulated by an external trigger. In the present work, two deactivatable bisubstrate inhibitors of basophilic protein kinases (PKs) were constructed by conjugating the pharmacophores via linkers that could be cleaved in response to external stimuli. The inhibitor ARC-2121 incorporated a photocleavable nitrodibenzofuran-comprising β-amino acid residue in the structure of the linker. The pharmacophores of the other deactivatable inhibitor ARC-2194 were conjugated via reduction-cleavable disulfide bond. The disassembly of the inhibitors was monitored by HPLC-MS. The affinity and inhibitory potency of the inhibitors toward cAMP-dependent PK (PKAcα) were established by an equilibrium competitive displacement assay and enzyme activity assay, respectively. The deactivatable inhibitors possessed remarkably high 1–2-picomolar affinity toward PKAcα. Irradiation of ARC-2121 with 365 nm UV radiation led to reaction products possessing a 30-fold reduced affinity. The chemical reduction of ARC-2194 resulted in the decrease of affinity of over four orders of magnitude. The deactivatable inhibitors of PKs are valuable tools for the temporal inhibition or capture of these pharmacologically important enzymes.

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.

Protein kinases catalyze phosphorylation, a small yet crucial modification that affects participation of the substrate proteins in the intracellular signaling pathways. The activity of 538 protein kinases encoded in human genome relies upon spatiotemporally controlled mechanisms, ensuring correct progression of virtually all physiological processes on the cellular level—from cell division to cell death. The aberrant functioning of protein kinases is linked to a wide spectrum of major health issues including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory diseases, etc. Hence, significant effort of scientific community has been dedicated to the dissection of protein kinase pathways in their natural milieu. The combination of recent advances in the field of light microscopy, the wide variety of genetically encoded or synthetic photoluminescent scaffolds, and the techniques for intracellular delivery of cargoes has enabled design of a plethora of probes that can report activation of target protein kinases in human live cells. The question remains: how much do we bias intracellular signaling of protein kinases by monitoring it? This review seeks answers to this question by analyzing different classes of probes according to their general structure, mechanism of recognition of biological target, and optical properties necessary for the reporting of intracellular events.