New Mitochondria-Targeted Fisetin Derivative Compromises Mitophagy and Limits Survival of Drug-Induced Senescent Breast Cancer Cells.

Mitochondria are considered as promising targets for cancer treatment. In the present study, triphenyl phosphonium cationic group-conjugated fisetin (mito-fisetin) was synthesized, and its anticancer activity was investigated in several cellular models of estrogen receptor (ER)-positive breast cancer in vitro and in vivo in proliferating and tamoxifen-promoted senescent states. Mito-fisetin, when used at low micromolar concentrations, stimulated the dissipation of mitochondrial membrane potential and oxidative stress, and affected mitochondrial function, resulting in apoptosis induction in senescent breast cancer cells. Mito-fisetin-mediated cytotoxicity was due to increased levels of phosphorylated AMPK, decreased levels of AKT and HSP90, and impaired mitophagic response, as judged by the analysis of the markers of mitophagosome formation. Senescent breast cancer cells were found to be more sensitive to mito-fisetin treatment than proliferating ones. We postulate that mitochondrial targeting in the case of fisetin may be considered as a promising anticancer and senotherapeutic strategy to eliminate drug-resistant senescent breast cancer cells.

Anticancer Activity of Encapsulated Pearl Millet Polyphenol-Rich Extract against Proliferating and Non-Proliferating Breast Cancer Cells In Vitro.

Plant-derived polyphenols are bioactive compounds with potential health-promoting properties including antioxidant, anti-inflammatory, and anticancer activity. However, their beneficial effects and biomedical applications may be limited due to their low bioavailability. In the present study, we have considered a microencapsulation-based drug delivery system to investigate the anticancer effects of polyphenol-rich (apigenin, caffeic acid, and luteolin) fractions, extracted from a cereal crop pearl millet (Pennisetum glaucum), using three phenotypically different cellular models of breast cancer in vitro, namely triple negative HCC1806, ER-positive HCC1428, and HER2-positive AU565 cells. Encapsulated polyphenolic extract induced apoptotic cell death in breast cancer cells with different receptor status, whereas it was ineffective against non-tumorigenic MCF10F cells. Encapsulated polyphenolic extract was also found to be cytotoxic against drug-resistant doxorubicin-induced senescent breast cancer cells that were accompanied by increased levels of apoptotic and necrotic markers, cell cycle inhibitor p21 and proinflammatory cytokine IL8. Furthermore, diverse responses to the stimulation with encapsulated polyphenolic extract in senescent breast cancer cells were observed, as in the encapsulated polyphenolic extract-treated non-proliferating AU565 cells, the autophagic pathway, here cytotoxic autophagy, was also induced, as judged by elevated levels of beclin-1 and LC3b. We show for the first time the anti-breast cancer activity of encapsulated polyphenolic extract of pearl millet and postulate that microencapsulation may be a useful approach for potentiating the anticancer effects of phytochemicals with limited bioavailability.

Carbon-Coated Iron Oxide Nanoparticles Promote Reductive Stress-Mediated Cytotoxic Autophagy in Drug-Induced Senescent Breast Cancer Cells.

The surface modification of magnetite nanoparticles (Fe3O4 NPs) is a promising approach to obtaining biocompatible and multifunctional nanoplatforms with numerous applications in biomedicine, for example, to fight cancer. However, little is known about the effects of Fe3O4 NP-associated reductive stress against cancer cells, especially against chemotherapy-induced drug-resistant senescent cancer cells. In the present study, Fe3O4 NPs in situ coated by dextran (Fe3O4@Dex) and glucosamine-based amorphous carbon coating (Fe3O4@aC) with potent reductive activity were characterized and tested against drug-induced senescent breast cancer cells (Hs 578T, BT-20, MDA-MB-468, and MDA-MB-175-VII cells). Fe3O4@aC caused a decrease in reactive oxygen species (ROS) production and an increase in the levels of antioxidant proteins FOXO3a, SOD1, and GPX4 that was accompanied by elevated levels of cell cycle inhibitors (p21, p27, and p57), proinflammatory (NFκB, IL-6, and IL-8) and autophagic (BECN1, LC3B) markers, nucleolar stress, and subsequent apoptotic cell death in etoposide-stimulated senescent breast cancer cells. Fe3O4@aC also promoted reductive stress-mediated cytotoxicity in nonsenescent breast cancer cells. We postulate that Fe3O4 NPs, in addition to their well-established hyperthermia and oxidative stress-mediated anticancer effects, can also be considered, if modified using amorphous carbon coating with reductive activity, as stimulators of reductive stress and cytotoxic effects in both senescent and nonsenescent breast cancer cells with different gene mutation statuses.

Mutation Status and Glucose Availability Affect the Response to Mitochondria-Targeted Quercetin Derivative in Breast Cancer Cells.

Mitochondria, the main cellular power stations, are important modulators of redox-sensitive signaling pathways that may determine cell survival and cell death decisions. As mitochondrial function is essential for tumorigenesis and cancer progression, mitochondrial targeting has been proposed as an attractive anticancer strategy. In the present study, three mitochondria-targeted quercetin derivatives (mitQ3, 5, and 7) were synthesized and tested against six breast cancer cell lines with different mutation and receptor status, namely ER-positive MCF-7, HER2-positive SK-BR-3, and four triple-negative (TNBC) cells, i.e., MDA-MB-231, MDA-MB-468, BT-20, and Hs 578T cells. In general, the mito-quercetin response was modulated by the mutation status. In contrast to unmodified quercetin, 1 µM mitQ7 induced apoptosis in breast cancer cells. In MCF-7 cells, mitQ7-mediated apoptosis was potentiated under glucose-depleted conditions and was accompanied by elevated mitochondrial superoxide production, while AMPK activation-based energetic stress was associated with the alkalization of intracellular milieu and increased levels of NSUN4. Mito-quercetin also eliminated doxorubicin-induced senescent breast cancer cells, which was accompanied by the depolarization of mitochondrial transmembrane potential. Limited glucose availability also sensitized doxorubicin-induced senescent breast cancer cells to apoptosis. In conclusion, we show an increased cytotoxicity of mitochondria-targeted quercetin derivatives compared to unmodified quercetin against breast cancer cells with different mutation status that can be potentiated by modulating glucose availability.

Electrospun fiber-based micro- and nano-system for delivery of high concentrated quercetin to cancer cells.

The anticancer potential of quercetin (Q), a plant-derived flavonoid, and underlining molecular mechanisms are widely documented in cellular models in vitro. However, biomedical applications of Q are limited due to its low bioavailability and hydrophilicity. In the present study, the electrospinning approach was used to obtain polylactide (PLA) and PLA and polyethylene oxide (PEO)-based micro- and nanofibers containing Q, namely PLA/Q and PLA/PEO/Q, respectively, in a form of non-woven fabrics. The structure and physico-chemical properties of Q-loaded fibers were characterized by scanning electron and atomic force microscopy (SEM and AFM), X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), goniometry and FTIR and Raman spectroscopy. The anticancer action of PLA/Q and PLA/PEO/Q was revealed using two types of cancer and nine cell lines, namely osteosarcoma (MG-63, U-2 OS, SaOS-2 cells) and breast cancer (SK-BR-3, MCF-7, MDA-MB-231, MDA-MB-468, Hs 578T, and BT-20 cells). The anticancer activity of Q-loaded fibers was more pronounced than the action of free Q. PLA/Q and PLA/PEO/Q promoted cell cycle arrest, oxidative stress and apoptotic cell death that was not overcome by heat shock protein (HSP)-mediated adaptive response. PLA/Q and PLA/PEO/Q were biocompatible and safe, as judged by in vitro testing using normal fibroblasts. We postulate that PLA/Q and PLA/PEO/Q with Q releasing activity can be considered as a novel and more efficient micro- and nano-system to deliver Q and eliminate phenotypically different cancer cells.

TRDMT1-mediated RNA C-5 methylation as a novel target in anticancer therapy.

Affected landscape of RNA modifications is frequently observed in different cancer cells that can be associated with the development of cancer cell phenotypic traits such as sustained proliferation, migration and invasion, apoptosis resistance and metabolic reprograming. DNMT2/TRDMT1 5-methylcytosine methyltransferase, initially classified as DNA methyltransferase, can methylate both tRNA and mRNA promoting tRNA stability and proper protein synthesis, and orchestrating DNA damage response (DDR) and DNA stability, respectively. TRDMT1 is associated with cancer progression as its levels can be elevated and its mutations can be observed in a number of cancer types. TRDMT1 gene knockout (KO) can sensitize cancer cells of different origin to radiotherapy and chemotherapy. In the present review paper, based on literature data, the physiological and pathophysiological roles of TRDMT1 in different biological systems are described with the emphasis on human normal and cancer cells. Potential TRDMT1 substrates, inhibitors and regulatory mechanisms of catalytic activity and cellular localization are also presented and evaluated. TRDMT1 as a novel promising target in anticancer therapy is proposed and discussed.

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells.

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Knockout of TRDMT1 methyltransferase affects DNA methylome in glioblastoma cells.

PURPOSE: We have previously shown that TRDMT1 methyltransferase is a regulator of chemotherapy-associated responses in glioblastoma cells. Despite the fact that glioblastoma, a common and malignant brain tumor, is widely characterized in terms of genetic and epigenetic markers, there are no data on TRDMT1-related changes in 5-methylcytosine pools in the genome. In the present study, the effect of TRDMT1 gene knockout (KO) on DNA methylome was analyzed. METHODS: CRISPR-based approach was used to obtain TRDMT1 KO glioblastoma cells. Total 5-methylcytosine levels in DNA, DNMT1 pools and DNMT activity were studied using ELISA. Reduced representation bisulfite sequencing (RRBS) was considered to comprehensively evaluate DNA methylome in glioblastoma cells with TRDMT1 KO. RESULTS: TRDMT1 KO cells were characterized by decreased levels of total 5-methylcytosine in DNA and DNMT1, and DNMT activity. RRBS-based methylome analysis revealed statistically significant differences in methylation-relevant DMS-linked genes in control cells compared to TRDMT1 KO cells. TRDMT1 KO-associated changes in DNA methylome may affect the activity of several processes and pathways such as telomere maintenance, cell cycle and longevity regulating pathway, proteostasis, DNA and RNA biology. CONCLUSIONS: TRDMT1 may be suggested as a novel modulator of gene expression by changes in DNA methylome that may affect cancer cell fates during chemotherapy. We postulate that the levels and mutation status of TRDMT1 should be considered as a prognostic marker and carefully monitored during glioblastoma progression.

The interplay between telomeric complex members and BCR::ABL1 oncogenic tyrosine kinase in the maintenance of telomere length in chronic myeloid leukemia.

PURPOSE: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by recurrent genetic aberration in leukemic stem cells, namely Philadelphia chromosome caused by reciprocal translocation t(9;22)(q34;q11). In our study, we analyzed the telomeric complex expression and function in the molecular pathogenesis of CML. METHODS: We employed CD34+ primary leukemic cells, comprising both leukemic stem and progenitor cell populations, isolated from peripheral blood or bone marrow of CML patients in chronic and blastic phase to analyze the telomere length and telomeric-associated proteins. RESULTS: The reduction in telomere length during disease progression was correlated with increased expression of BCR::ABL1 transcript and the dynamic changes were neither associated with the enzymatic activity of telomerase nor with gene copy number and expression of telomerase subunits. Increased expression of BCR::ABL1 was positively correlated with expression of TRF2, RAP1, TPP1, DKC1, TNKS1, and TNKS2 genes. CONCLUSIONS: The dynamics of telomere length changes in CD34+ CML cells is dependent on the expression level of BCR::ABL, which promotes the expression of certain shelterins including RAP1 and TRF2, as well as TNKS, and TNKS2, and results in telomere shortening regardless of telomerase activity. Our results may allow better understanding of the mechanisms responsible for the genomic instability of leukemic cells and CML progression.

Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS.

Many neurodegenerative disorders are characterized by the abnormal aggregation of misfolded proteins that form amyloid deposits which possess prion-like behavior such as self-replication, intercellular transmission, and consequent induction of native forms of the same protein in surrounding cells. The distribution of the accumulated proteins and their correlated toxicity seem to be involved in the progression of nervous system degeneration. Molecular chaperones are known to maintain proteostasis, contribute to protein refolding to protect their function, and eliminate fatally misfolded proteins, prohibiting harmful effects. However, chaperone network efficiency declines during aging, prompting the onset and the development of neurological disorders. Extracellular vesicles (EVs) are tiny membranous structures produced by a wide range of cells under physiological and pathological conditions, suggesting their significant role in fundamental processes particularly in cellular communication. They modulate the behavior of nearby and distant cells through their biological cargo. In the pathological context, EVs transport disease-causing entities, including prions, α-syn, and tau, helping to spread damage to non-affected areas and accelerating the progression of neurodegeneration. However, EVs are considered effective for delivering therapeutic factors to the nervous system, since they are capable of crossing the blood-brain barrier (BBB) and are involved in the transportation of a variety of cellular entities. Here, we review the neurodegeneration process caused mainly by the inefficiency of chaperone systems as well as EV performance in neuropathies, their potential as diagnostic biomarkers and a promising EV-based therapeutic approach.

DNMT2/TRDMT1 gene knockout compromises doxorubicin-induced unfolded protein response and sensitizes cancer cells to ER stress-induced apoptosis.

The acidic, hypoxic and nutrient-deprived tumor microenvironment may induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) may exert an important cytoprotective role by promoting folding of newly synthesized proteins and cancer cell survival. The lack of DNMT2/TRDMT1 methyltransferase-mediated C38 tRNA methylation compromises translational fidelity that may result in the accumulation of misfolded and aggregated proteins leading to proteotoxic stress-related cell death. In the present study, DNMT2/TRDMT1 gene knockout-mediated effects were investigated during doxorubicin (DOX)-induced ER stress and PERK-, IRE1- and ATF6-orchestrated UPR in four genetically different cellular models of cancer (breast and cervical cancer, osteosarcoma and glioblastoma cells). Upon DOX stimulation, DNMT2/TRDMT1 gene knockout impaired PERK activation and modulated NSUN and 5-methylcytosine RNA-based responses and microRNA profiles. The lack of DNMT2/TRDMT1 gene in DOX-treated four cancer cell lines resulted in decreased levels of four microRNAs, namely, miR-23a-3p, miR-93-5p, miR-125a-5p and miR-191-5p involved in the regulation of several pathways such as ubiquitin-mediated proteolysis, amino acid degradation and translational misregulation in cancer. We conclude that DNMT2/TRDMT1 gene knockout, at least in selected cellular cancer models, affects adaptive responses associated with protein homeostasis networks that during prolonged ER stress may result in increased sensitivity to apoptotic cell death.

Non-targeting siRNA-mediated responses are associated with apoptosis in chemotherapy-induced senescent skin cancer cells.

It is widely accepted that siRNA transfection can promote some off-target effects in the genome; however, little is known about how the cells can respond to the presence of non-viral dsRNA. In the present study, non-targeting control siRNA (NTC-siRNA) was used to evaluate its effects on the activity of pathogen and host-derived nucleic acid-associated signaling pathways such as cGAS-STING, RIG-I, MDA5 and NF-κB in A431 skin cancer cells and BJ fibroblasts. NTC-siRNA treatment promoted cytotoxicity in cancer cells. Furthermore, NTC-siRNA-treated doxorubicin-induced senescent cancer cells were more prone to apoptotic cell death compared to untreated doxorubicin-induced senescent cancer cells. NTC-siRNA stimulated the levels of NF-κB, APOBECs, ALY, LRP8 and phosphorylated STING that suggested the involvement of selected components of nucleic acid sensing pathways in NTC-siRNA-mediated cell death response in skin cancer cells. NTC-siRNA-mediated apoptosis in cancer cells was not associated with IFN-ß-based pro-inflammatory response and TRDMT1-based adaptive response. In contrast, in NTC-siRNA-treated fibroblasts, an increase in the levels of RIG-I and IFN-ß was not accompanied by affected cell viability. We propose that the use of NTC-siRNA in genetic engineering may provoke a number of unexpected effects that should be carefully monitored. In our experimental settings, NTC-siRNA promoted the elimination of doxorubicin-induced senescent cancer cells that may have implications in skin cancer therapies.

Synergic Temperature Effect of Star-like Monodisperse Iron Oxide Nanoparticles and Their Related Responses in Normal and Cancer Cells.

Magnetic nanoparticle (MNP) anisotropy has been tailored by the preparation of MNPs having different shapes (star-like, cubic, and polyhedral) using a self-modified rapid hot-injection process. The surface modification of MNPs was performed through etidronic ligand grafting with a strong binding affinity to mixed metal oxides, ensuring sufficient colloidal stability, surface protection, and minimized aggregation and interparticle interactions. The heating effect was induced by contactless external stimulation through the action of an alternating magnetic field and NIR laser radiation (808 nm). The efficacy of the energy conversion was evaluated as a function of the particle shape, concentration, and external stimuli parameters. In turn, the most efficient star-like particles have been selected to study their response in contact with normal and cancer cells. It was found that the star-like MNPs (Fe3O4 SL-NPs) at 2 mg/mL concentration induce necrosis and significantly alter cell cycle progression, while 0.5 mg/mL can stimulate the antioxidative and anti-inflammatory response in normal cells. A biologically relevant heating effect leading to heat-mediated cell death was achieved at a 2 mg/mL concentration of star-like particles and was enhanced by the addition of ascorbic acid (AA). AA-mediated photomagnetic hyperthermia can lead to the modulation of the heat-shock response in cancer cells that depends on the genotypic and phenotypic variations of cell lines.

Perspectives on using bacteriophages in biogerontology research and interventions.

With the development of materials engineering, gerontology-related research on new tools for diagnostic and therapeutic applications, including precision and personalised medicine, has expanded significantly. Using nanotechnology, drugs can be precisely delivered to organs, tissues, cells, and cell organelles, thereby enhancing their therapeutic effects. Here, we discuss the possible use of bacteriophages as nanocarriers that can improve the safety, efficiency, and sensitivity of conventional medical therapies. Phages are a new class of targeted-delivery vectors, which can carry high concentrations of cargo and protect other nontargeted cells from the senescent cell killing effects of senolytics. Bacteriophages can also be subjected to chemical and/or genetic modifications that would acquire novel properties and improve their ability to detect senescent cells and deliver senolytics. Phage research in experimental biogerontology will also develop strategies to efficiently deliver senolytics, target senescent cells, activate extrinsic apoptosis pathways in senescent cells, trigger immune cells to recognise senescent cells, induce autophagy, promote cell and tissue regeneration, inhibit senescence-associated secretory phenotype (SASP) by senomorphic activity, stimulate the properties of mild stress-inducing hormetic agents and hormetins, and modulate the gut microbiome.

RNA 5-methylcytosine status is associated with DNMT2/TRDMT1 nuclear localization in osteosarcoma cell lines.

Osteosarcoma (OS) is a pediatric malignant bone tumor with unsatisfying improvements in survival rates due to limited understanding of OS biology and potentially druggable targets. The present study aims to better characterize osteosarcoma U-2 OS, SaOS-2, and MG-63 cell lines that are commonly used as in vitro models of OS. We focused on evaluating the differences in cell death pathways, redox equilibrium, the activity of proliferation-related signaling pathways, DNA damage response, telomere maintenance, DNMT2/TRDMT1-based responses and RNA 5-methylcytosine status. SaOS-2 cells were characterized by higher levels of superoxide and nitric oxide that promoted AKT and ERK1/2 activation thus modulating cell death pathways. OS cell lines also differed in the levels and localization of DNA repair regulator DNMT2/TRDMT1. SaOS-2 cells possessed the lowest levels of total, cytoplasmic and nuclear DNMT2/TRDMT1, whereas in MG-63 cells, the highest levels of nuclear DNMT2/TRDMT1 were associated with the most pronounced status of RNA 5-methylcytosine. In silico analysis revealed potential phosphorylation sites at DNMT2/TRDMT1 that may be related to the regulation of DNMT2/TRDMT1 localization. We postulate that redox homeostasis, proliferation-related pathways and DNMT2/TRDMT1-based effects can be modulated as a part of anti-osteosarcoma strategy reflecting diverse phenotypic features of OS cells.

Lapatinib- and fulvestrant-PAMAM dendrimer conjugates promote apoptosis in chemotherapy-induced senescent breast cancer cells with different receptor status.

Lapatinib (L) and fulvestrant (F) are used in targeted anticancer therapies, in particular, against phenotypically different breast cancer cells. L, a dual inhibitor of EGFR and HER2 tyrosine kinases, is active against HER2-positive breast cancer cells, while F, a selective estrogen receptor degrader (SERD), is active against ER-positive breast cancer cells. However, the action of L and F can be limited due to their relatively low water solubility and bioavailability. In the present study, poly(amidoamine) (PAMAM) dendrimer G3 was functionalized with L or F or L and F to compare their effects with free L or F against breast cancer cells with different receptor status (ER-positive MCF-7, triple negative MDA-MB-231 and HER2-positive SK-BR-3 cells). L-PAMAM and F-PAMAM conjugates potentiated cytostatic and cytotoxic action of L and F that was accompanied by elevated levels of autophagy. TRDMT1, RNA methyltransferase, was also involved in this response as judged by TRDMT1 nuclear translocation and nano-drug resistance of TRDMT1 gene knockout cells. Nano-drugs also promoted elimination of doxorubicin-induced senescent breast cancer cells by apoptosis-mediated senolysis regardless of receptor status. In conclusion, we propose a novel anticancer approach based on L-PAMAM and F-PAMAM nanoplatforms being effective, at least, against breast cancer cells with different phenotypic features.

Optical Control of Tissue Regeneration through Photostimulation of Organic Semiconducting Nanoparticles.

Next generation bioengineering strives to identify crucial cues that trigger regeneration of damaged tissues, and to control the cells that execute these programs with biomaterials and devices. Molecular and biophysical mechanisms driving embryogenesis may inspire novel tools to reactivate developmental programs in situ. Here nanoparticles based on conjugated polymers are employed for optical control of regenerating tissues by using an animal with unlimited regenerative potential, the polyp Hydra, as in vivo model, and human keratinocytes as an in vitro model to investigate skin repair. By integrating animal, cellular, molecular, and biochemical approaches, nanoparticles based on poly-3-hexylthiophene (P3HT) are shown able to enhance regeneration kinetics, stem cell proliferation, and biomolecule oxidation levels. Opposite outputs are obtained with PCPDTBT-NPs (Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b'] dithiophene)-alt-4,7(2,1,3-benzothiadiazole)], causing a beneficial effect on Hydra regeneration but not on the migratory capability of keratinocytes. These results suggest that the artificial modulation of the redox potential in injured tissues may represent a powerful modality to control their regenerative potential. Importantly, the possibility to fine-tuning materials' photocatalytic efficiency may enable a biphasic modulation over a wide dynamic range, which can be exploited to augment the tissue regenerative capacity or inhibit the unlimited potential of cancerous cells in pathological contexts.

Analysis of Mutational Profile of Hypopharyngeal and Laryngeal Head and Neck Squamous Cell Carcinomas Identifies KMT2C as a Potential Tumor Suppressor.

Hypopharyngeal cancer is a poorly characterized type of head and neck squamous cell carcinoma (HNSCC) with bleak prognosis and only few studies focusing specifically on the genomic profile of this type of cancer. We performed molecular profiling of 48 HPV (Human Papilloma Virus)-negative tumor samples including 23 originating from the hypopharynx and 25 from the larynx using a targeted next-generation sequencing approach. Among genes previously described as significantly mutated, TP53, FAT1, NOTCH1, KMT2C, and CDKN2A were found to be most frequently mutated. We also found that more than three-quarters of our patients harbored candidate actionable or prognostic alterations in genes belonging to RTK/ERK/PI3K, cell-cycle, and DNA-damage repair pathways. Using previously published data we compared 67 hypopharyngeal cancers to 595 HNSCC from other sites and found no prominent differences in mutational frequency except for CASP8 and HRAS genes. Since we observed relatively frequent mutations of KTM2C (MLL3) in our dataset, we analyzed their role, in vitro, by generating a KMT2C-mutant hypopharyngeal cancer cell line FaDu with CRISPR-Cas9. We demonstrated that KMT2C loss-of-function mutations resulted in increased colony formation and proliferation, in concordance with previously published results. In summary, our results show that the mutational profile of hypopharyngeal cancers might be similar to the one observed for other head and neck cancers with respect to minor differences and includes multiple candidate actionable and prognostic genetic alterations. We also demonstrated, for the first time, that the KMT2C gene may play a role of tumor suppressor in HNSCC, which opens new possibilities in the search for new targeted treatment approaches.

5-Azacytidine Inhibits the Activation of Senescence Program and Promotes Cytotoxic Autophagy during Trdmt1-Mediated Oxidative Stress Response in Insulinoma ß-TC-6 Cells.

5-Azacytidine (5-azaC), a methyltransferase inhibitor and anticancer drug, can promote several cellular stress responses such as apoptosis, autophagy, and senescence. The action of 5-azaC is complex and can be modulated by dose, time of treatment, and co-administration with oxidants. Insulinoma is a rare pancreatic neuroendocrine tumor with limited chemotherapeutic options. In the present study, two cellular models of insulinoma were considered, namely NIT-1 and ß-TC-6 mouse cells, to evaluate the effects of 5-azaC post-treatment during hydrogen peroxide-induced oxidative stress. 5-azaC attenuated the development of oxidant-induced senescent phenotype in both cell lines. No pro-apoptotic action of 5-azaC was observed in cells treated with the oxidant. On the contrary, 5-azaC stimulated an autophagic response, as demonstrated by the increase in phosphorylated eIF2α and elevated pools of autophagic marker LC3B in oxidant-treated ß-TC-6 cells. Notably, autophagy resulted in increased necrotic cell death in ß-TC-6 cells with higher levels of nitric oxide compared to less affected NIT-1 cells. In addition, 5-azaC increased levels of RNA methyltransferase Trdmt1, but lowered 5-mC and m6A levels, suggesting Trdmt1 inhibition. We postulate that the 5-azaC anticancer action may be potentiated during oxidative stress conditions that can be used to sensitize cancer cells, at least insulinoma cells, with limited drug responsiveness.