Normal Diploid Fibroblasts Research Articles

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

Evaluation of the Cytotoxic, Antioxidative and Antimicrobial Effects of Dracocephalum moldavica L. Cultivars.

The aim of the present study was to correlate the antioxidant, antimicrobial, and cytotoxic activities of hydroalcoholic extracts obtained from the aerial parts of three Dracocephalum moldavica L. cultivars with their polyphenolic compositions. The polyphenols were identified and quantified using spectrophotometrical methods and LC-MS analysis. Their antioxidant capacities were assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. Their in vitro antimicrobial efficacies were assessed using the agar well diffusion and broth microdilution methods. Their cytotoxicity was investigated on normal diploid foreskin fibroblasts (BJ) and on colorectal adenocarcinoma (DLD-1) cell lines. The results pointed out significant amounts of polyphenolic compounds in the compositions of the tested cultivars, with rosmarinic acid as the main compound (amounts ranging between 5.337 ± 0.0411 and 6.320 ± 0.0535 mg/mL). All three cultivars displayed significant antioxidant (IC50 ranging between 35.542 ± 0.043 and 40.901 ± 0.161 µg/mL for the DPPH assay, and for the FRAP assay 293.194 ± 0.213 and 330.165 ± 0.754 µmol Trolox equivalent/mg dry vegetal material) and antimicrobial potential (especially towards the Gram-positive bacteria), as well as a selective toxicity towards the tumoral line. A significant positive correlation was found between antioxidant activity and the total phenolic acids (r2 = 0.987) and polyphenols (r2 = 0.951). These findings bring further arguments for strongly considering D. moldavica cultivars as promising vegetal products, which warrants further investigation.

Exonuclease 1 Drives Growth and Genomic Instability in Multiple Myeloma through Interaction with CAD, a Key Protein Involved in De Novo Pyrimidine Biosynthesis

Human exonuclease 1 (Exo1) plays important roles in DNA replication and repair including the resection of DNA ends during homologous recombination (HR). Based on our previous data demonstrating the role of HR in genomic instability and growth of cancer cells, we investigated role of EXO1 in multiple myeloma (MM). Expression of EXO1 and pRPA32 (a marker of DNA end resection) was elevated in all myeloma cell lines tested (N=10) relative to normal peripheral blood mononuclear (PBMC) cell samples. We developed a fluorescence-based EXO1 activity assay and observed elevated (~1.5- to > 5-fold; p < 0.05) Exo1 activity in all twelve MM cell lines tested relative to normal PBMCs. Moreover., elevated expression of EXO1 correlated with poor overall and event-free survival in MM patients (MMRF dataset; p < 1x10-5). Transgenic suppression of EXO1 in MM cell lines caused a time-dependent loss in cell viability. Evaluation at day 5 after selection indicated that, relative to control cells, EXO1 suppression in RPMI, MM1S, MM1R and U266 cells was associated with ∼95%, 85%, 60% and 40% cell death, respectively. Apoptosis, as evaluated by annexin-labeling, was detected in ~50% of EXO1-knockdown cells compared to 11.9% of control MM1S cells. Conditional knockdown in H929 cells evaluation at 48 h after adding doxycycline demonstrated apoptosis in 18% of EXO1-suppressed cells vs. 4.9% of control cells. To investigate the mechanisms by which EXO1 impacts MM cell viability, the proteins interacting with EXO1 in MM cell lines (MM1S and H929) were identified using Exo1 pull-down and mass spectrometry. Interaction of EXO1 with CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a trifunctional protein involved in the first three steps of de novo pyrimidine biosynthesis, was identified and its binding confirmed with co-immunoprecipitation. We found that EXO1 overexpression increased the growth rate of non-cancerous cell types (HS5 - bone marrow stromal cells and normal fibroblasts), whereas suppression of CAD inhibited the spontaneous and EXO1-mediated increase in growth rate as well as DNA replication in both normal cell types. Consistently, the CAD-knockdown increased apoptosis in HS5 cells by ∼5-fold, whereas EXO1 overexpression reduced it by ~2-fold. Conversely, suppression of CAD also increased apoptosis in EXO1-overexpressing cells by 3-fold. In normal diploid fibroblast cells with a limited lifespan, EXO1 overexpression increased DNA replication by 32%, whereas CAD-knockdown reduced it by ∼65%. Importantly, CAD-knockdown in EXO1-overexpressing cells also reduced DNA replication by 26%. These data indicate that CAD is involved in EXO1-dependent cell proliferation and survival. Investigating the molecular events associated with EXO1, we observed that its suppression in MM cells inhibits DNA end resection, DNA breaks, HR activity and genomic instability (as assessed by a micronucleus assay); whereas overexpression in normal cells increased those activities. Moreover, inhibition of CAD reduced spontaneous as well as EXO1-induced HR activity and genomic instability in both hematopoietic and solid tumor cell types. Since CAD is involved in the first three steps of pyrimidine biosynthesis, our data suggest that interaction with CAD enables EXO1 to drive DNA replication, HR and associated genomic instability. Functional relevance of this interaction and possible regulation of de novo pyrimidine biosynthesis by EXO1 is being further investigated. Inhibitors of CAD may provide a novel therapeutic target in myeloma as well as other Exo1 overexpressing malignancies.

Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts.

Mitochondria are dysfunctional in post-senescent cells. Therefore, age-dependent impairment of mitochondrial energy production accompanied by excessive mitochondrial reactive oxygen species (ROS) is proposed to be a key driver of cellular senescence, which is a state of irreversible cell cycle arrest. However, it remains to be clarified whether mitochondrial dysfunction initiates or accelerates replicative senescence. In this study, we observed no increase in mitochondrial ROS or decrease in mitochondrial respiratory function in human TIG-1 fibroblasts in the transition phase, during which the population doubling rate gradually decreases due to the development of replicative senescence. The integrated stress response and expression of growth differentiation factor 15, which are triggered by respiratory chain deficiency, were also not induced in the transition phase. Mitochondria were elongated without aberrant cristae structures in the transition phase. Mitophagy-related protein levels started to decrease in the transition phase, but autophagic flux slightly increased during replicative senescence. These results suggest that mitochondrial dysfunction and excessive mitochondrial ROS generation do not occur predominately in the transition phase and may not play a role in the development of replicative senescence in normal diploid TIG-1 fibroblasts.

Cytotoxic Alkylynols of the Sponge Cribrochalina vasculum: Structure, Synthetic Analogs and SAR Studies.

A series of twenty-three linear and branched chain mono acetylene lipids were isolated from the Caribbean Sea sponge Cribrochalina vasculum. Seventeen of the compounds, 1–17, are new, while six, 18–23, were previously characterized from the same sponge. Some of the new acetylene-3-hydroxy alkanes 1, 6, 7, 8, 10 were tested for selective cytotoxicity in non-small cell lung carcinoma (NSCLC) cells over WI-38 normal diploid lung fibroblasts. Compound 7, presented clear tumor selective activity while, 1 and 8, showed selectivity at lower doses and 6 and 10, were not active towards NSCLC cells at all. The earlier reported selective cytotoxicity of some acetylene-3-hydroxy alkanes (scal-18 and 23), in NSCLC cells and/or other tumor cell types were also confirmed for 19, 20 and 22. To further study the structure activity relationships (SAR) of this group of compounds, we synthesized several derivatives of acetylene-3-hydroxy alkanes, rac-18, scal-S-18, R-18, rac-27, rac-32, R-32, S-32, rac-33, rac-41, rac-42, rac-43, rac-45, rac-48 and rac-49, along with other 3-substituted derivatives, rac-35, rac-36, rac-37, rac-38, rac-39 and rac-40, and assessed their cytotoxic activity against NSCLC cells and diploid fibroblasts. SAR studies revealed that the alcohol moiety at position 3 and its absolute R configuration both were essential for the tumor cell line selective activity while for its cytotoxic magnitude the alkyl chain length and branching were of less significance.

Aurora A Inhibitor TAS-119 Enhances Antitumor Efficacy of Taxanes In Vitro and In Vivo: Preclinical Studies as Guidance for Clinical Development and Trial Design.

TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro, TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines. Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5. In vivo, TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors. Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel. The degree of enhancement calculated as fold of change of the IC50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration. The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later. This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination.

A screening program on anti-metastasis from microbial metabolites

A malignant tumor is hard to eradicate by removing the mass surgically because the abnormal cells often metastasize. How to find and kill these metastatic cells is an important key to cure cancer. We have isolated over 100 filamentous fungi from soil of extreme environments in Taiwan, including forest, hot spring, lake and fruit tree rhizosphere in order to screen some biologically active compounds. Screening of anti-metastatic compounds, we used a set of highly metastatic cell lines, B16-F10 (mouse, melanoma) and HT-1080 (human, fibrosarcoma) and cell lines with low metastatic ability, B 16-F1 (mouse, melanoma) and MRC-5 (human, lung, normal diploid fibroblast). Cultures grown in microtiter plates were assayed by SRB (sulforhodamine B) method. The goal for this screening program is to isolate biologically active compounds that can inhibit the growth of highly metastatic cells such as B16-F10 and HT-1080 but little effect on low metastatic cells such as B10-F1 and MRC-5. Some strains' media showed significant differences in cytotoxicity between the highly metastatic cells and the low metastatic cells. These results suggest that some products in these media may contain metastasis-specific or cell-specific toxins.

Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype.

Primary diploid cells exit the cell cycle in response to exogenous stress or oncogene activation through a process known as cellular senescence. This cell-autonomous tumor-suppressive mechanism is also a major mechanism operative in organismal aging. To date, temporal aspects of senescence remain understudied. Therefore, we use quantitative proteomics to investigate changes following forced HRASG12V expression and induction of senescence across 1 week in normal diploid fibroblasts. We demonstrate that global intracellular proteomic changes correlate with the emergence of the senescence-associated secretory phenotype and the switch to robust cell cycle exit. The senescence secretome reinforces cell cycle exit, yet is largely detrimental to tissue homeostasis. Previous studies of secretomes rely on ELISA, bottom-up proteomics or RNA-seq. To date, no study to date has examined the proteoform complexity of secretomes to elucidate isoform-specific, post-translational modifications or regulated cleavage of signal peptides. Therefore, we use a quantitative top-down proteomics approach to define the molecular complexity of secreted proteins <30 kDa. We identify multiple forms of immune regulators with known activities and affinities such as distinct forms of interleukin-8, as well as GROα and HMGA1, and temporally resolve secreted proteoform dynamics. Together, our work demonstrates the complexity of the secretome past individual protein accessions and provides motivation for further proteoform-resolved measurements of the secretome.

PFG acted as an inducer of premature senescence in TIG-1 normal diploid fibroblast and an inhibitor of mitosis in the HeLa cells.

Our previous work has reported an anti-proliferative compound from moutan cortex, paeoniflorigenone which can induce cancer-selective apoptosis. However, its anti-proliferative mechanism is still unknown. According to morphology changes (hypertrophy and flattening), we hypothesized that PFG can induce senescence or inhibit cell mitosis. Here we show that PFG can induce cellular senescence, evidenced by the expression of senescence-associated β-galactosidase, G0/G1 cell cycle arrest and permanent loss of proliferative ability, in normal TIG-1 diploid fibroblast but not cancerous HeLa cells. In cancerous HeLa cells, PFG inhibited proliferation by inducing S and G2/M cell cycle arrest and mitosis inhibition. DNA damage response was activated by PFG, interestingly the reactive oxygen species level was suppressed instead of escalated. To sum up, we report 3 new roles of PFG as, 1. inducer of premature senescence in normal TIG-1 cells, 2. inhibitor of mitosis in cancerous HeLa cells, 3. ROS scavenger. Abbreviations: PFG: Paeoniflorigenone; ROS: reactive oxygen species; ATM: ataxia telangiectasia mutated; t-BHP: tert-butyl hydroperoxide; SA-β-gal: senescence-associatedβ-galactosidase; DNA-PKcs: DNA-dependent protein kinase; γ-H2AX: H2AX phosphoryla-tion at Ser-139.

In Vitro Evaluation of Chitosan Induced Cytotoxicity against Cancerous Cell lines: MCF-7, Saos-2 and HeLa

Objective/ Background: Cancer is still the most common cause of morbidity in world and new powerful anticancer agents without severe side effects from natural sources is important. Methods: The evaluation of cytotoxicity and apoptosis induction was carried out in MCF-7,HeLa and Saos-2 as cancerous cell lines with different histological origin and human fibroblast served as control normal cell. The cells were treated with different concentrations of chitosan and the cytotoxicity was determined using MTT assay after 24, 48 and 72 h .The mode of death was evaluated by flow cytometry . Results: While both types of chitosan showed significant concentration-dependently cytotoxic effects against the three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. On the other hand, there were no significant differences between LMWC and HMWC cytotoxicity in all cell lines. The flow cytometry results showed the apoptosis pattern of death more in Saos-2 and HeLa while necrosis was more observable with MCF7. Also higher viability with both types of chitosan was seen in fibroblast as normal cells Conclusion: Chitosan shows anticancerous effect against 3 cancerous cell lines, while it is compatible with normal diploid fibroblast cells. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

Anticancer properties of chitosan against osteosarcoma, breast cancer and cervical cancer cell lines.

Background:Cancer is still the most common cause of morbidity in the world. Chitosan, a commonly used natural polymer, is consisted of different molecular weight with different biological activities.The purpose of this study was to determine cytotoxicity effect of high molecular weight (HMWC) and low molecular weight of chitosan (LMWC) on three cancerous cell lines MCF-7, HeLa and Saos-2 with different histological origin.Methods:The anticancer property of two types of chitosan on three cancerous cell lines and human fibroblast as normal cell line, was evaluated by cytotoxic activity including their apoptosis induction properties. Chitosan solutions 2% (w/v) were prepared. The cells were treated by different concentration of chitosan and viability was determined by MTT assay after 24, 48 and 72 h .Also the mode of cell death-apoptosis vs necrosis ,was determined by Annexin V staining assay and analyzed by flow cytometry.Results:While both types of chitosan were effective in inhibiting cell proliferation of three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. Despite of a significant decrease in all 3 cell lines viability, up to 90%, but we didn't see a concentration dependent difference between both types of chitosan (HMWC and LMWC) in their cytotoxic effects. Flow cytometry analysis showed necrosis more observable with MCF7 while the apoptosis pattern of death was more in Saos-2 and HeLa. Also, higher viability with both types of chitosan was seen in fibroblast as normal cells.Conclusion:While chitosan is compatible with normal diploid fibroblast cells, it shows anticancerous effect against 3 cancerous cell lines. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

On Choosing Control Objects in Experimental Gerontological Research

Recently, a large number of papers have appeared that describe the successful use of various biologically active compounds (short peptides, mitochondrial antioxidants, antidiabetic biguanides, mimetics of dietary restriction, autophagy modulators, etc.) as geroprotectors. However, in our opinion, in most cases, the positive results of such studies are determined by a “successful” selection of control objects. Animals with certain abnormalities are often used for this purpose, so that any favorable effect on the corresponding pathological processes leads to an increase in their lifespan. In addition, control animals can be normal (i.e., wildtype) but placed under certain extreme conditions that can be overcome just by using certain biologically active compounds. Thus, in this case, the treatment of pathologies rather than the effect on fundamental processes of aging is observed. There is a point of view that the results of Clive McCay’s well-known experiments, which have significantly prolonged the life of rats by limiting caloric intake, were determined by the facts that, firstly, the control animals fed ad libitum (which is absolutely untypical for animals in the wild) and, secondly, Fisher-344 rats, which were used in these experiments, are short-lived. The above considerations, apparently, also apply to the gerontological experiments on cultured cells. In particular, we sometimes hear remarks from our colleagues regarding the model of “stationary phase aging” of cell cultures, which is used in our laboratory, due to the fact that most of the experiments are performed on transformed rather than normal cells. However, this approach seems to us quite justified, because the phenomenon of “stationary phase”/chronological aging is common to a wide variety of cells, including bacteria, yeasts, cyanobacteria, mycoplasmas, as well as animal and plant cells. Cells with an unlimited mitotic potential do not change either from experiment to experiment or during long-term cultivation both with and without subcultivation (within the framework of the stationary phase aging model), which cannot be said of the normal diploid fibroblasts, whose telomeres are shortened with each division. In the period from seeding to entering the stationary phase of growth, the cells divide up to ten times! We believe that, to search for effective geroprotectors that affect the fundamental mechanisms of aging, it is necessary to perform studies on “maximally healthy” animals or on “maximally stable” model systems.

Evaluation of the Antioxidant Capacity, Antimicrobial and Antiproliferative Potential of Fir (Abies alba Mill.) Honeydew Honey Collected from Gorski kotar (Croatia).

SUMMARYThe paper examines the antiproliferative, antimicrobial and antioxidative effects of fir (Abies alba Mill.) honeydew honey from mountain region of Croatia (Gorski kotar) as a potential replacement for standard antibiotics and chemotherapeutic agents. Cell viability, annexin V assay and flow cytometry analysis served to analyse the antiproliferative effect on, apoptosis induction in and cell death of cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 and normal diploid human fibroblasts (BJ). Antimicrobial activity was tested against Staphylococcus and Acinetobacter strains by agar well diffusion and microdilution assays. The DPPH˙ assay determined the radical scavenging activity, while mathematical models helped to evaluate the kinetic data of DPPH˙ inhibition. Antiproliferative effect on all tested cell lines and the prominent effect on normal diploid human fibroblasts (BJ), colorectal adenocarcinoma (SW620, metastatic) and breast epithelial adenocarcinoma (MCF-7, metastatic) was observable. The mechanisms of antiproliferative effect included accumulation of cells in the sub-G1 phase in all tested cells and induction of apoptosis in SW620 and MCF-7 cells predominantly. The antibacterial assays showed that antibiotic-resistant strains of both bacteria, including multi-resistant strain A. baumannii ATCC® BAA-1605™, were sensitive to all tested honey samples. Radical scavenging assay suggests that antioxidants present in the honey possess different radical suppressing abilities and that they react at different rates with radicals, thereby causing two steps of reaction. The results of the study indicate that Croatian fir honeydew honey has a therapeutic potential due to the strong biological activity and can serve to protect human health.

Nuclear insulin-like growth factor-1 receptor (IGF1R) displays proliferative and regulatory activities in non-malignant cells.

The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF1 and IGF2. The IGF1R is involved in both physiological and pathological activities and is usually overexpressed in most types of cancer. In addition to its classical mechanism of action, recent evidence has shown a nuclear presence of IGF1R, associated with novel genomic/transcriptional types of activities. The present study was aimed at evaluating the hypothesis that nuclear IGF1R localization is not restricted to cancer cells and might constitute a novel physiologically relevant regulatory mechanism. Our data shows that nuclear translocation takes place in a wide array of cells, including normal diploid fibroblasts. In addition, we provide evidence for a synergistic effect of a nuclear translocation blocker along with selective IGF1R inhibitors in terms of decreasing cell proliferation. Given the important role of the IGF1R in mitogenesis, the present results may be of translational relevance in cancer research. In conclusion, results are consistent with the concept that nuclear IGF1R fulfills important physiological and pathological roles.

In vitro evaluation of the potential toxic effects of palladium nanoparticles on fibroblasts and lung epithelial cells

Palladium nanoparticles have been increasingly used in catalytic processes, wastewater treatment, electronics, and biomedicine. However, recent evidence proved that these nanoparticles are able to induce adverse effects both in in vitro and in vivo models. Nevertheless, molecular mechanisms underlying the toxic effects are still poorly understood. Therefore, this study aimed to investigate the potential toxicological mechanisms of palladium nanoparticles assessing their effects on normal diploid rat fibroblast and lung carcinoma human epithelial cell lines. Several endpoints such as cell growth, cell cycle progression, DNA damage, induction of apoptosis, reactive oxygen species production and expression of cell cycle regulatory proteins were evaluated. Results showed that palladium nanoparticles inhibited cell growth in a dose- and time-dependent manner in both cell lines, although with a more evident action on fibroblasts. Interestingly, inhibition of cell growth was not associated with the induction of apoptosis. Cell cycle progression was arrested in the G0/G1 phase and DNA damage was evident in both cell lines even if only a slight increase in the intracellular reactive oxygen species levels was detected. These findings provide valuable insight into understanding the molecular mechanisms responsible of palladium nanoparticles toxicity whose identification is essential to define an adequate risk assessment process.

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert's membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

Compounds from the marine sponge Cribrochalina vasculum offer a way to target IGF-1R mediated signaling in tumor cells.

In this work two acetylene alcohols, compound 1 and compound 2, which were isolated and identified from the sponge Cribrochalina vasculum, and which showed anti-tumor effects were further studied with respect to targets and action mechanisms. Gene expression analyses suggested insulin like growth factor receptor (IGF-1R) signaling to be instrumental in controlling anti-tumor efficacy of these compounds in non-small cell lung cancer (NSCLC). Indeed compounds 1 and 2 inhibited phosphorylation of IGF-1Rβ as well as reduced its target signaling molecules IRS-1 and PDK1 allowing inhibition of pro-survival signaling. In silico docking indicated that compound 1 binds to the kinase domain of IGF-1R at the same binding site as the well known tyrosine kinase inhibitor AG1024. Indeed, cellular thermal shift assay (CETSA) confirmed that C. vasculum compound 1 binds to IGF-1R but not to the membrane localized tyrosine kinase receptor EGFR. Importantly, we demonstrate that compound 1 causes IGF-1Rβ but not Insulin Receptor degradation specifically in tumor cells with no effects seen in normal diploid fibroblasts. Thus, these compounds hold potential as novel therapeutic agents targeting IGF-1R signaling for anti-tumor treatment.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Disruption of Direct 3-Dimensional (3D) Telomere-TRF2 (Telomere Related Factor 2) Interaction Is a Hallmark of Primary Hodgkin (H) and Reed-Sternberg (RS) Cells

Introduction: Numerical and complex structural chromosomal aberrations are regularly identified in primary mononuclear H and multinucleated RS cells and in Hodgkin's lymphoma (HL) derived H cell lines. The transition from H to RS cells is associated with dynamic progressive 3D telomere dysfunction, major changes in the telomere-protecting shelterin complex and formation of giant "zebra" chromosomes. Analogous findings are observed in an in vitro model for post-germinal center B-cell, Epstein-Barr-virus (EBV)-associated HL (Blood. 2015;125 (13):2101-2110). In this experimental system the EBV encoded oncogene LMP1 mediates multinuclearity through downregulation of TRF2. Thus, we hypothesized that the 3D interaction of telomeres and TRF2 is directly associated with the formation of H and RS cells. To this end, we developed a combined quantitative 3D TRF2-telomere immuno FISH protocol (3D TRF2/Telo-Q-FISH) and applied it to monolayers of primary H and RS cells enriched from diagnostic lymph node biopsies allowing 3D analysis of the entire nuclear content (often not achieved using laser microdissection of H and RS cells, given that their nuclei are generally >10 um in diameter and that this technique is performed on 5 um sections).Methods: 3D TRF2/Telo-Q-FISH(Blood. 2015;125 (13):2101-2110; data supplement) was performed on cultured BJ-5ta normal diploid fibroblasts and B-cell rich suspensions (negative selection) of diagnostic lymph nodes, cultured for 12-36 hours. Quality of H and RS cells was assessed by Wright-Giemsa staining. Lymphocytes, H and RS cells of 10 classical HL were analyzed, 3 of them containing LMP1+ tumor cells (EBV-associated HL).Results: Whereas the BJ-5ta normal diploid control fibroblasts and most internal control lymphocytes showed quantitatively and qualitatively intact 1:1, tight physical 3D association of TRF2 with telomeres, with only few very short telomeres (<20%), the H and RS cells of all 10 cases consistently displayed telomere-TRF2 steric disruption, with a significant increase of very short telomeres (49-71%). Interestingly, two opposite disruption patterns of the 3D telomere-TRF2 interaction were identified.The 3 LMP1+ cases were characterized by abundant telomeres without TRF2 interaction (de-protected). TRF2 spots were rare and the quantitative ratio of telomere/TRF2 signals was > 6:1. This loss of TRF2 expression showed gradual progression from H to RS cells.The 7 LMP1- cases showed a different 3D telomere-TRF2 pattern. Three cases with clinically aggressive disease (stages IVA, IVB and IIIB) were characterized by abundant TRF2 signals, most of them not associated with telomeres. In two of them huge multinucleated RS "ghost" cells without any or only few telomeres but numerous TRF2 signals and internuclear DNA-bridges were observed. In one of these two and in the third case bi-nucleated RS cells with grossly different nuclear telomere content of nucleus one and nucleus two were present. Most importantly, these 3 cases showed an inversion of the quantitative ratio of telomere/TRF2 signals <1:3 and the progressive loss of telomere signals from H to RS cells. The remaining 4 cases had a mixed pattern with an overall moderate loss of TRF2 signals including TRF2 de-protected telomeres but also TRF2 signals not associated with telomeres (quantitative, telomere/TRF2 signals ratio of about 1.5-2:1).Conclusions: Disruption of the 3D TRF2-telomere interaction was demonstrated in 100% of primary H and RS cells. However, two opposite pathogenetic mechanisms appear to be operative: in EBV-associated HL telomere de-protection is due to an impressive loss of TRF2 signals physically linked to telomeres, in agreement with the hypothesis of our in vitro model for EBV-associated HL. In clinically aggressive EBV-negative HL massive attrition of telomere signals is associated with an overwhelming increase in TRF2 signals that are not associated with telomeres. In both scenarios, the telomeres are predominantly very short and the crucial 3D interaction is disrupted, thereby qualifying HL as a shelterin-associated disease with the telomere-shelterin complex playing the role of a "plaque tournante" in the pathogenesis of HL. Further molecular dissection of the shelterin-telomere interaction may offer new therapeutic targets for refractory/relapsing disease. DisclosuresNo relevant conflicts of interest to declare.

MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.