Murine Embryonic Fibroblasts Research Articles

BUBR1 deficiency results in abnormal megakaryopoiesis

AbstractThe physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1–/– embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1+/– blastocysts grew relatively normally in vitro, BUBR1–/– blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1+/– mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference–mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1+/– mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis.

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents.

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).

FUBP1 and FUBP2 enforce distinct epigenetic setpoints for MYC expression in primary single murine cells

Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular “cruise controls” that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.

Essential Roles of Cohesin STAG2 in Mouse Embryonic Development and Adult Tissue Homeostasis

Cohesin mediates sister chromatid cohesion and 3D genome folding. Two versions of the complex carrying STAG1 or STAG2 coexist in somatic vertebrate cells. STAG2 is commonly mutated in cancer, and germline mutations have been identified in cohesinopathy patients. To better understand the underlying pathogenic mechanisms, we report the consequences of Stag2 ablation in mice. STAG2 is largely dispensable in adults, and its tissue-wide inactivation does not lead to tumors but reduces fitness and affects both hematopoiesis and intestinal homeostasis. STAG2 is also dispensable for murine embryonic fibroblasts invitro. In contrast, Stag2-null embryos die by mid-gestation and show global developmental delay and defective heart morphogenesis, most prominently in structures derived from secondary heart field progenitors. Both decreased proliferation and altered transcription of tissue-specific genes contribute to these defects. Our results provide compelling evidence on cell- and tissue-specific roles of different cohesin complexes and how their dysfunction contributes to disease.

P53 Disruption Increases Uracil Accumulation in DNA of Murine Embryonic Fibroblasts and Leads to Folic Acid–Nonresponsive Neural Tube Defects in Mice

Neural tube defects (NTDs) occur in nervous tissue during embryogenesis when the neural tube fails to close. Approximately 70% of all human NTDs can be prevented by folic acid (FA). Altered expression and/or function of the tumor suppressor protein p53 can lead to NTDs in mouse models. The aim of this study was to determine if dietary FA could rescue p53-/--induced NTDs in mice, and to determine the effect loss of p53 has on pathways in folate 1-carbon metabolism. p53+/- female mice were randomly allocated and weaned onto either an FA-sufficient diet (2mg/kg folic acid; +FA), or an FA-deficient diet (-FA). After 8 wk, the females were time-mated to p53-/- males. Embryos were examined at E12.5 for NTDs. Folate enzyme concentrations, nucleotide synthesis, uracil accumulation in DNA, and proliferation were measured in primary murine embryonic fibroblasts (MEFs). The "n - 1" chi-square test was used to compare NTD percentages, whereas all other data were analyzed by Student t test, except where noted a multilevel-fit model was used. NTD rates of litters from dams consuming the +FA diet (20/46; 43%) did not differ from those of litters from dams consuming the -FA diet (14/35; 40%) (P>0.05). p53-/- MEFs had 55% higher rates of folate-dependent de novo dTMP synthesis, a ∼2-fold higher accumulation of uracil in DNA, and a ∼30% higher rate of proliferation (P≤0.05) than p53+/- MEFs independent of folate. p53-related NTDs are not FA responsive. Increased dTMP synthesis in p53-/- MEFs might not have been sufficient to meet the demands for thymidine triphosphate (dTTP) synthesis as evidenced by the elevated amounts of uracil in DNA. This study provides additional evidence that elevated uracil in DNA is a risk factor for NTDs.

KLF3 Mediates Epidermal Differentiation through the Epigenomic Writer CBP.

SummaryImpairments in the differentiation process can lead to skin diseases that can afflict ∼20% of the population. Thus, it is of utmost importance to understand the factors that promote the differentiation process. Here we identify the transcription factor KLF3 as a regulator of epidermal differentiation. Knockdown of KLF3 results in reduced differentiation gene expression and increased cell cycle gene expression. Over half of KLF3's genomic binding sites occur at active enhancers. KLF3 binds to active enhancers proximal to differentiation genes that are dependent upon KLF3 for expression. KLF3's genomic binding sites also highly overlaps with CBP, a histone acetyltransferase necessary for activating enhancers. Depletion of KLF3 causes reduced CBP localization at enhancers proximal to differentiation gene clusters, which leads to loss of enhancer activation but not priming. Our results suggest that KLF3 is necessary to recruit CBP to activate enhancers and drive epidermal differentiation gene expression.

ERRATUM.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

The LUBAC participates in lysophosphatidic acid-induced NF-κB activation

The natural bioactive glycerophospholipid lysophosphatidic acid (LPA) binds to its cognate G protein-coupled receptors (GPCRs) on the cell surface to promote the activation of several transcription factors, including NF-κB. LPA-mediated activation of NF-κB relies on the formation of a signalosome that contains the scaffold CARMA3, the adaptor BCL10 and the paracaspase MALT1 (CBM complex). The CBM complex has been extensively studied in lymphocytes, where it links antigen receptors to NF-κB activation via the recruitment of the linear ubiquitin assembly complex (LUBAC), a tripartite complex of HOIP, HOIL1 and SHARPIN. Moreover, MALT1 cleaves the LUBAC subunit HOIL1 to further enhance NF-κB activation. However, the contribution of the LUBAC downstream of GPCRs has not been investigated. By using murine embryonic fibroblasts from mice deficient for HOIP, HOIL1 and SHARPIN, we report that the LUBAC is crucial for the activation of NF-κB in response to LPA. Further echoing the situation in lymphocytes, LPA unbridles the protease activity of MALT1, which cleaves HOIL1 at the Arginine 165. The expression of a MALT1-insensitive version of HOIL1 reveals that this processing is involved in the optimal production of the NF-κB target cytokine interleukin-6. Lastly, we provide evidence that the guanine exchange factor GEF-H1 favors MALT1-mediated cleavage of HOIL1 and NF-κB signaling in this context. Together, our results unveil a critical role for the LUBAC as a positive regulator of NF-κB signaling downstream of LPA receptors.

The role of the adaptor molecule STING during Schistosoma mansoni infection

Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting−/− and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting−/− mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting−/− animals. Surprisingly, Sting−/− mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting−/− neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting−/− mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.

Adipose triglyceride lipase activity regulates cancer cell proliferation via AMP-kinase and mTOR signaling

Aberrant fatty acid (FA) metabolism is a hallmark of proliferating cells, including untransformed fibroblasts or cancer cells. Lipolysis of intracellular triglyceride (TG) stores by adipose triglyceride lipase (ATGL) provides an important source of FAs serving as energy substrates, signaling molecules, and precursors for membrane lipids. To investigate if ATGL-mediated lipolysis impacts cell proliferation, we modified ATGL activity in murine embryonic fibroblasts (MEFs) and in five different cancer cell lines to determine the consequences on cell growth and metabolism. Genetic or pharmacological inhibition of ATGL in MEFs causes impaired FA oxidation, decreased ROS production, and a substrate switch from FA to glucose leading to decreased AMPK-mTOR signaling and higher cell proliferation rates. ATGL expression in these cancer cells is low when compared to MEFs. Additional ATGL knockdown in cancer cells did not significantly affect cellular lipid metabolism or cell proliferation whereas the ectopic overexpression of ATGL increased lipolysis and reduced proliferation. In contrast to ATGL silencing, pharmacological inhibition of ATGL by Atglistatin© impeded the proliferation of diverse cancer cell lines, which points at an ATGL-independent effect. Our data indicate a crucial role of ATGL-mediated lipolysis in the regulation of cell proliferation. The observed low ATGL activity in cancer cells may represent an evolutionary selection process and mechanism to sustain high cell proliferation rates. As the increasing ATGL activity decelerates proliferation of five different cancer cell lines this may represent a novel therapeutic strategy to counteract uncontrolled cell growth.

Abstract B53: Differential response of distinct KRAS mutants to SHP2 inhibition

Abstract Introduction: RAS is mutated in 30% of tumors and is still considered “undruggable.” Among RAS genes KRAS is the most frequently mutated, mostly at residues G12, G13 and Q61, impairing its intrinsic and GAP-mediated GTP hydrolysis capacity (Prior et al., Cancer Res 2012). Nevertheless, specific mutations are differently distributed in human cancers (G12 89%, G13 9%, Q61 1%). In particular, G12D (36%) is prevalent over G12C (14%) and G12V (23%). Distinct oncogenic mutations differently affect RAS GTP hydrolysis capacity (Lu et al., Sci Rep 2016; Hunter et al., Mol Cancer Research 2015), with G12C preserving the highest GTPase activity and Q61 mutants being the most compromised. This implies that some KRAS mutants could be sensitive to upstream regulation, as suggested by the in vivo sensitivity of KRAS mutant tumors to SHP2 inhibitors (Mainardi et al. and Ruess et al., Nat Med 2018; Fedele et al., Cancer Discov 2018). In order to finely predict the response to the SHP2 inhibitor SHP099 (Chen et al., Nature 2016) in KRAS mutant patients, we used a panel of isogenic Rasless murine embryonic fibroblasts (MEFs) (Esposito et al., Semin Cancer Biol 2018) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes, and studied their response to SHP099, either alone or in combination with the MEK inhibitor AZD6244. Materials and Method: The panel of isogenic RAS-dependent MEFs was provided by the NCI RAS Initiative. In vitro, RAS-GTP loading was evaluated and dose-response curves generated based on cell viability assays. For xenograft experiments, 5x106 cells were subcutaneously injected in NSG mice. After tumor establishment (200mm3) mice were treated either with vehicle, SHP099 75mg/kg, AZD6244 25 mg/Kg or a combination of the last two. Results and Discussion: In vitro, we found that when cultured in 3% FCS, the most GTPase-proficient KRAS mutants (G13D and G12C) are more sensitive to SHP099 as compared to the most impaired (G12D and Q61R). Response to SHP099 was accompanied by a reduction in RAS-GTP loading. Surprisingly, in vivo results using xenograft models showed no correlation between residual GTPase activity of the mutants and sensitivity to SHP099. Nevertheless, synergy between SHP2 and MEK inhibitors treatment was more pronounced in the GTPase-proficent mutants, suggesting a possible correlation between residual intrinsic GTP hydrolysis capacity of the mutants and their resistance to MEK inhibitors through feedback activation of receptor tyrosine kinases (Sun et al., Cell Rep 2014). Conclusion: Our data confirm single-agent activity of SHP099 against proliferation driven by a subset of KRAS mutants, and suggest that mutants with higher residual GTPase capacity could better benefit from combined SHP2 and MEK inhibition. Citation Format: Sara Mainardi, Astrid Bosma, Antonio Mulero-Sánchez, René Bernards. Differential response of distinct KRAS mutants to SHP2 inhibition [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B53.

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models.

Human liver models that are three-dimensional (3D) in architecture are indispensable for compound metabolism/toxicity screening, to model liver diseases for drug discovery, and for cell-based therapies; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months. Therefore, here we determined how microscale 3D collagen I presentation and fibroblast interaction affect the longevity of PHHs. High-throughput droplet microfluidics was utilized to generate reproducibly sized (∼300-μm diameter) microtissues containing PHHs encapsulated in collagen I ± supportive fibroblasts, namely, 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as controls. Hepatic functions and gene expression were subsequently measured for up to 6 weeks. We found that microtissues placed within multiwell plates rescued PHH functions at 2- to 30-fold higher levels than spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Importantly, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically relevant manner to drug-mediated cytochrome P450 induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display high hepatic functions for 6+ weeks and are useful for assessing drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find utility for modeling liver diseases and as building blocks for cell-based therapies.

PTEN inhibitor VO-OHpic suppresses TSC2- / - MEFs proliferation by excessively inhibiting autophagy via the PTEN/PRAS40 pathway.

Tuberous sclerosis complex (TSC) is a relatively rare autosomal dominant disease which involves multiple organs, including the brain, kidney, lung, skin and heart. Renal angiomyolipomas (RAML) are the main causes of mortality in patients with TSC. The preferred treatment for RAML is the use of mTOR inhibitors, but the efficacy of these are not satisfactory. Therefore, an alternative treatment is urgently required. Autophagy levels decline in TSC associated cortical tubers, and the inhibition of autophagy in animal or cell models of TSC may suppress tumor development and cell proliferation. PTEN is a protein tyrosine phosphatase and can inhibit the activation of Akt. In the present study, it was indicated that the PTEN inhibitor, hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid (VO-OHpic), suppressed proliferation and growth of TSC2-/- murine embryonic fibroblasts (MEFs) by further inhibiting autophagy of cells. The expression levels of human microtubule-associated protein 1 light chain 3-I (LC3-I) and LC3-II, which are autophagy associated proteins, were demonstrated to decline following VO-OHpic treatment. The expression levels of phosphorylated proline-rich Akt substrate 40 kDa (PRAS40) also decreased in TSC2-/- MEFs treated with VO-OHpic. The PTEN inhibitor may inhibit the proliferation of TSC2-/- MEFs through the PTEN-PRAS40 pathway by excessively inhibiting autophagy, without the dependence of the Ras homolog, mTORC1 binding/mTOR pathway. PTEN may be a potential therapeutic target for the treatment of TSC. Further in vivo studies are required to confirm these results.

Glutathione S-transferase P influences the Nrf2-dependent response of cellular thiols to seleno-compounds.

Recent findings suggest a functional interaction of the drug resistance enzyme glutathione S-transferase P (GSTP) with the transcription factor Nrf2, a master regulator of the adaptive stress response to cellular electrophiles. The effect of this interaction on the metabolism and redox of cellular thiols was investigated in this studyduring the exposure to alkylating Se-compounds in murine embryonic fibroblasts (MEFs). GSTP1-1 gene ablation was confirmed to upregulate Nrf2 activity and to increase Cys uptake and the de novo biosynthesis of reduced glutathione (GSH)that was readily released in the extracellular medium together with other cellular thiols. This latter response was associated with a higher expression of the membrane transporter MRP1 and was markedly stimulated by the treatment with alkylating Se-compounds together with protein S-glutathionylation that was observed to be under the influence of GSTP expression. The response of cellular thiols to Se-compounds was not altered by the transient (SiRNA-induced) or stable inactivation of NRF2 in GSTP competent or hGSTP1 transfected cells, while defects of GSH biosynthesis, efflux, and redox were observed after NRF2 silencing in GSTP-/- MEFs. In conclusion, GSTP is confirmed to functionally interact with Nrf2 and to have a prominent position in the pecking order of factors that control both the Nrf2-dependent and independent response of cellular thiols to alkylating agents.

Abstract P4-08-03: Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer

Abstract Research Overview and Objective Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. Methods To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. Results Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53−/−ARF−/− mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. Implication/Discussion These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures. Citation Format: Che-Pei Kung, Catherine E Kuzmicki, Emily A Bross, Sua Ryu, Eric Freeman, Thwisha Sabloak, Emily R Bramel, Leonard B Maggi Jr, Jason D Weber. Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-03.

Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II.

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation.

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (109 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.

Silencing of PARP2 Blocks Autophagic Degradation.

Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.

A simple procedure to obtain nanodiamonds from leftover of HFCVD system for biological application

Nanodiamonds (NDs) are amongst the most investigated carbon-based nanostructures due to their chemical stability and favorable mechanical properties. Despite the number of works on methods for NDs production, one of the main challenges is to achieve their colloidal stability in aqueous suspension. Additionally, NDs are normally obtained by expensive, complex and time-consuming process. Herein, it was presented a facile method to obtain NDs in aqueous suspension by using columnar structure diamond from Hot-Filament Chemical Vapour Deposition reactor (HFCVD). CVD diamond leftover thick film from CVDVale Company was used. Therefore, this method has the advantage of being not only practical but also cost-effective since it brings a profitable use of CVD diamond leftover. The Diamond thick film was submitted to ultrasonic cavitation in the presence and absence of ZrO2 microbeads in aqueous medium. The NDs hydrodynamic diameter and the stability in aqueous suspension were monitored by light scattering, size and morphology were analyzed by transmission electronic microscopy. Considering the wide application of NDs in biomedical devices, cytotoxicity of aqueous suspensions of NDs was evaluated against murine embryonic fibroblast cells. Furthermore, NDs were functionalized with hydrogen and carboxyl groups. NDs aqueous suspension of straight size distribution was obtained even in the absence of ZrO2 beads, indicating that they may be dispensable in order to decrease NDs size. NDs of average hydrodynamic diameter of 22 nm and − 35 mV of Zeta-potential were obtained after ultrasonic cavitation followed by 2 h of centrifugation, not demonstrating cytotoxicity to cells at very low (0.05–0.5 μg/mL) nor at higher concentrations (116 μg/mL). Nevertheless, NDs showed a moderate cytotoxicity at intermediary concentration range (0.5–2.2 μg/mL). From our knowledge, this is the first work that reports on a facile method for providing NDs aqueous suspension with high colloidal stability from HFCVD diamond leftover.