Frentizole derivatives with mTOR inhibiting and senomorphic properties.

Frentizole is immunosuppressive drug with low acute toxicity and lifespan-prolonging effect. Recently, frentizole´s potential to disrupt toxic amyloid ß (Aß) - Aß-binding alcohol dehydrogenase (ABAD) interaction in mitochondria in Alzheimer´s brains has been revealed. Another broadly studied drug with anti-aging and immunosuppressive properties is an mTOR inhibitor - rapamycin. Since we do not yet precisely know what is behind the lifespan-prolonging effect of rapamycin and frentizole, whether it is the ability to inhibit the mTOR signaling pathway, reduction in mitochondrial toxicity, immunosuppressive effect, or a combination of all of them, we have decided within our previous work to dock the entire in-house library of almost 240 Aß-ABAD modulators into the FKBP-rapamycin-binding (FRB) domain of mTOR in order to interlink mTOR-centric and mitochondrial free radical-centric theories of aging and thus to increase the chances of success. Based on the results of the docking study, molecular dynamic simulation and MM-PBSA calculations, we have selected nine frentizole-like compounds (1 - 9). Subsequently, we have determined their real physical-chemical properties (logP, logD, pKa and solubility in water and buffer), cytotoxic/cytostatic, mTOR inhibitory, and in vitro anti-senescence (senolytic and senomorphic) effects. Finally, the three best candidates (4, 8, and 9) have been forwarded for in vivo safety studies to assess their acute toxicity and pharmacokinetic properties. Based on obtained results, only compound 4 demonstrated the best results within in vitro testing, the ability to cross the blood-brain barrier and the lowest acute toxicity (LD50 in male mice 559 mg/kg; LD50 in female mice 575 mg/kg).

Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

STAT3 inhibitor Stattic and its analogues inhibit STAT3 phosphorylation and modulate cytokine secretion in senescent tumour cells.

Signal transducer and activator of transcription 3 (STAT3) signalling serves an important role in carcinogenesis and cellular senescence, and its inhibition in tumour cells represents an attractive therapeutic target. Premature cellular senescence, a process of permanent proliferative arrest of cells in response to various inducers, such as cytostatic drugs or ionizing radiation, is accompanied by morphological and secretory changes, and by altered susceptibility to chemotherapeutic agents, which can thereby complicate their eradication by cancer therapies. In the present study, the responsiveness of proliferating and docetaxel (DTX)­induced senescent cancer cells to small molecule STAT3 inhibitor Stattic and its analogues was evaluated using tumour cell lines. These agents displayed cytotoxic effects in cell viability assays on both proliferating and senescent murine TRAMP­C2 and TC­1 cells; however, senescent cells were markedly more resistant. Western blot analysis revealed that Stattic and its analogues effectively inhibited constitutive STAT3 phosphorylation in both proliferating and senescent cells. Furthermore, whether the Stattic­derived inhibitor K1836 could affect senescence induction or modulate the phenotype of senescent cells was evaluated. K1836 treatment demonstrated no effect on senescence induction by DTX. However, the K1836 compound significantly modulated secretion of certain cytokines (interleukin­6, growth­regulated oncogene α and monocyte chemoattractant protein­1). In summary, the present study demonstrated differences between proliferating and senescent tumour cells in terms of their susceptibility to STAT3 inhibitors and demonstrated the ability of the new STAT3 inhibitor K1836 to affect the secretion of essential components of the senescence­associated secretory phenotype. The present study may be useful for further development of STAT3 inhibitor­based therapy of cancer or age­related diseases.

SILAC-IodoTMT for Assessment of the Cellular Proteome and Its Redox Status.

Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).

Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics.

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

Synergism of BCL-2 family inhibitors facilitates selective elimination of senescent cells.

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics. Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells. Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

Phospho-SIM and exon8b of PML protein regulate formation of doxorubicin-induced rDNA-PML compartment.

Repetitive sequences are among the most unstable regions in the eukaryotic genome and defects in their maintenance correlate with premature aging and cancer development. Promyelocytic leukemia protein (PML) induces accumulation of proteins at distinct nuclear sites, thereby affecting a plethora of processes including DNA repair or maintenance of telomeres. Doxorubicin, the broadly used chemotherapeutic compound, induces formation of PML-nucleolar associations (PNAs). Nevertheless, molecular factors affecting formation of PNAs are still largely unknown. Here we show that PNAs can accumulate ribosomal DNA (rDNA) and, after restoration of RNA polymerase I activity, these structures transfer a fraction of rDNA outside the nucleolus. Mutagenesis of PML isoforms revealed that this process depends on the SUMO-interacting motif and adjacent serine-rich region, and is enhanced by exon8b present exclusively in PML IV isoform. Moreover, we demonstrate that PNAs formation is also regulated by p14ARF/p53 tumor suppressors and casein kinase 2. Our data elucidate how PML nucleolar compartment is assembled, bring the first evidence of PML interacting with rDNA, and show the PML-dependent translocation of rDNA away from the nucleolus.

Peroxiredoxin 6 protects irradiated cells from oxidative stress and shapes their senescence-associated cytokine landscape.

Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

The Effect of Chemical Structure of OEG Ligand Shells with Quaternary Ammonium Moiety on the Colloidal Stabilization, Cellular Uptake and Photothermal Stability of Gold Nanorods.

PURPOSE: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain - (16-mercaptohexadecyl)trimethylammonium bromide (MTAB). METHODS: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation. RESULTS: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of OEG+GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm2) of femtosecond laser irradiation than the alkanethiol MTAB. CONCLUSION: OEG+GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.

Design, synthesis, and in vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition.

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure-activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.

Suprabasin-A Review.

Among the ~22,000 human genes, very few remain that have unknown functions. One such example is suprabasin (SBSN). Originally described as a component of the cornified envelope, the function of stratified epithelia-expressed SBSN is unknown. Both the lack of knowledge about the gene role under physiological conditions and the emerging link of SBSN to various human diseases, including cancer, attract research interest. The association of SBSN expression with poor prognosis of patients suffering from oesophageal carcinoma, glioblastoma multiforme, and myelodysplastic syndromes suggests that SBSN may play a role in human tumourigenesis. Three SBSN isoforms code for the secreted proteins with putative function as signalling molecules, yet with poorly described effects. In this first review about SBSN, we summarised the current knowledge accumulated since its original description, and we discuss the potential mechanisms and roles of SBSN in both physiology and pathology.

Aberrantly elevated suprabasin in the bone marrow as a candidate biomarker of advanced disease state in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto-oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid-derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon-gamma and demethylating agent 5-azacytidine (5-AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high-risk MDS with a possible role in disease progression and therapy resistance.

Isoform-resolved correlation analysis between mRNA abundance regulation and protein level degradation.

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli.

Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.

Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity.

The photothermal cancer therapy using cationic gold nanorods (GNRs) stabilized by quaternary ammonium salts (QAS) have a great potential to enhance conventional cancer treatment as it promises the effective eradication of cancer cells including cells resistant to radio- and chemo-therapy and the stimulation of anti-tumor immune response. However, as the cytotoxicity of the conventional alkanethiol-QAS compounds limits their utility in medicine, here we developed GNRs modified by novel highly hydrophilic cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain N,N,N-trimethyl-3,6,9,12,15-pentaoxaheptadecyl-17-sulfanyl-1-ammonium bromide (POSAB) showing insignificant cytotoxicity in the free state. Surface modification of GNRs by POSAB allowed to prepare nanoparticles with good stability in water, high cellular uptake and localization in lysosomes that are a promising alternative to alkanethiol-stabilized GNRs especially for biomedical applications.

Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology.

Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.

PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair.

The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair.

Interferon-regulated suprabasin is essential for stress-induced stem-like cell conversion and therapy resistance of human malignancies.

Radiation and chemotherapy represent standard-of-care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio- and chemotherapy, we exposed human cancer cell lines (HeLa, MCF-7 and DU145) to clinically relevant doses of 5-azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low-adherent stem-like cells. Stress-mobilized low-adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon-gamma but not interferon-beta resulted in the development of a heterogeneous, low-adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem-cell markers. Chemical inhibition of mitogen-activated protein kinase/ERK kinase (MEK) or siRNA-mediated knockdown of extracellular signal-regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low-adherent population, indicating that interferon-gamma-mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin-specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low-adherent cancer cells induced by 5-azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA-mediated knockdown of SBSN suppressed the low-adherent fraction in irradiated, interferon-gamma- and 5-azacytidine-treated cells, respectively, implicating SBSN in genotoxic stress-induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.

Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram.

BACKGROUND: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover. METHODS: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting. RESULTS: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155. CONCLUSIONS: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.