Subsequent Cell Growth Research Articles

Corynoxine exerts the anti-tumor effect on esophageal squamous cell carcinoma principally via the EZH2-DUSP5-ERK1/2-mediated cell growth inhibition

BackgroundEsophageal cancer is one of the most prevalent malignant tumors and the sixth largest cause of tumor-associated death worldwide. Squamous cell carcinoma (ESCC) accounts for 85 % of all esophageal cancer cases. ESCC treatment remains to be significantly difficult. Corynoxine (Cory) is a tetracyclic hydroxyindole alkaloid isolated from Uncaria macrophylla. It is unclear whether Cory has an anti-tumor effect on ESCC. PurposeTo determine the anti-tumor activity of Cory and the associated mechanisms in ESCC. Study designCory's effects on proliferation, apoptosis, migration, and invasion, as well as the underlying molecular causes were assessed using two ESCC cell lines, KYSE150 and TE-1. A xenograft mouse model was then applied to evaluate the anti-tumor activity of Cory in vivo. MethodsWestern blot, assays including CCK-8, colony formation, EdU staining, TUNEL staining, cell scratch and Transwell, and a xenograft mouse model were used in this study. ResultsCory suppressed cell growth, provoked cell apoptosis, and hindered cell migration and invasion of ESCC cells. DUSP5 knockdown reduced the Cory-induced cell death and restored cell migration and invasion through ERK1/2 activation. Further analyses showed that Cory promoted DUSP5 expression via inhibiting EZH2 expression, leading to inactivation of ERK1/2 signaling and the subsequent cell growth inhibition of ESCC. In vivo experiments disclosed that Cory suppressed tumor growth of ESCC through upregulating DUSP5 expression. ConclusionsCory plays an anti-tumor role in ESCC by regulating EZH2-DUSP5-ERK1/2 signaling pathway. Cory may be promising to be a novel therapy for treating ESCC.

Discovery of Potent and Selective Covalent Inhibitors of HER2WT and HER2YVMA.

HER2 overexpression and amplification have been identified as oncogenic drivers, and the development of therapies to treat tumors harboring these markers has received considerable attention. Activation of HER2 signaling and subsequent cell growth can also be induced by HER2 mutations, including the common YVMA insertion in exon 20 within the kinase domain. Enhertu is currently the only approved treatment for HER2 mutant tumors in NSCLC. TKIs tested in this space have suffered from off-target activity, primarily due to EGFRWT inhibition or attenuated activity against HER2 mutants. The goal of this work was to identify a TKI that would provide robust inhibition of oncogenic HER2WT and HER2 mutants while sparing EGFRWT activity. Herein, we describe the development of a potent, covalent inhibitor of HER2WT and the YVMA insertion mutant while providing oral bioavailability and avoiding the inhibition of EGFRWT.

Searching for novel MDM2/MDMX dual inhibitors through a drug repurposing approach

Disruption of p53-MDM2/MDMX interaction by smaller inhibitors is a promising therapeutic intervention gaining tremendous interest. However, no MDM2/MDMX inhibitors have been marketed so far. Drug repurposing is a validated, practical approach to drug discovery. In this regard, we employed structure-based virtual screening in a reservoir of marketed drugs and identified nintedanib as a new MDM2/MDMX dual inhibitor. The computational structure analysis and biochemical experiments uncover that nintedanib binds MDM2/MDMX similarly to RO2443, a dual MDM2/MDMX inhibitor. Furthermore, the mechanistic study reveals that nintedanib disrupts the physical interaction of p53-MDM2/MDMX, enabling the transcriptional activation of p53 and the subsequent cell cycle arrest and growth inhibition in p53+/+ cancer cells. Lastly, structural minimisation of nintedanib yields H3 with the equivalent potency. In summary, this work provides a solid foundation for reshaping nintedanib as a valuable lead compound for the further design of MDM2/MDMX dual inhibitors.

Second cell growth optimized by heating mode during two-step supercritical CO2 foaming polymer process

The batch foaming of supercritical fluids faces numerous challenges, such as poor controllability due to dynamic temperature changes during depressurization foaming, and prolonged saturation time and heterogeneous temperature distribution during temperature rising foaming, which leads to lower productivity and non-uniform cells. To overcome these challenges, a unique two-step foaming method in combination with microwave enhancement was proposed. The two-step foaming method involved depressurization foaming for cell nucleation and initial growth, followed by temperature rising foaming for subsequent cell growth, producing foams with higher expansion ratio. Meanwhile, a conduction-microwave (C-M) heating mechanism which integrates different heating schemes to enhance heating efficiency and uniformity was innovatively proposed. The effect of the microwave radiation time, power, initial environmental temperature, and content of CO2 in polymer on the foaming by C-M heating was studied. Compared to existing methods, the foaming by C-M heating produces polystyrene (PS) foam with lower density and more uniform cells.

Effective Chlorella vulgaris Biomass Harvesting through Sulfate and Chloride Flocculants

Efficient microalgae harvesting is a great challenge hindering diverse industrial applications of microalgae. Flocculation is regarded as an effective and promising technology for microalgae harvesting. In this study, sulfate (Al2(SO4)3 and Fe2(SO4)3) and chloride flocculants (AlCl3 and FeCl3) were used to harvest Chlorella vulgaris. Flocculation conditions, including flocculant dose, flocculation time, stirring speed, stirring time, and flocculation pH, were optimized, and flocculant effects on microalgal cell status, floc characteristics, biomass composition, algal cell re-culture, and media recycling were investigated. All flocculants exhibited efficient flocculation efficiency (93.5–98.8%) with lower doses of sulfate salts (60 mg/L algal culture) and higher doses of chloride salts (100 mg/L algal culture). The tested flocculants had no obvious influence on biomass composition (including lipids, carbohydrates, proteins, and carotenoids), and microalgal cells in flocs could efficiently regrow. The spent medium of all treatments was successfully recycled for subsequent cell growth, thus reducing dependency on fresh medium.

Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Abstract 2492: Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma

Abstract Hepatoblastoma represents the most common primary malignancy of the liver in childhood. Pathological activation of the WNT signaling pathway is characteristic for hepatoblastoma caused by mutations of CTNNB1 or other genes encoding components of the pathway. Genome-wide chromosomal analyses uncovered recurrent chromosomal alterations, in particular gain of chromosome 2q, and in some cases amplification of the region 2q24 that suggests the presence of a so far unidentified oncogene in this chromosomal region. The aim of this study was to identify and further characterize this oncogene. In the framework of the clinical hepatoblastoma study of the German Society for Pediatric Oncology and Hematology, we had the opportunity to study samples of 76 hepatoblastoma patients. Using molecular inversion probe (MIP) array technology, genome-wide, high-resolution, quantitative chromosomal copy number profiles were generated and the smallest overlapping amplified region was identified. RNA and protein expression in normal and diseased tissues as well as ascribed functions of the genes located in this area were evaluated in silico in available databases. RNA expression levels of these genes were analyzed by Nanostring method and Affymetrix arrays and compared between 2q24-amplified tumors versus non-amplified tumors and normal liver tissue. The most promising candidate gene regarding its expression pattern and described function was selected for further analysis. Its protein expression was studied in situ by immunohistochemistry in hepatoblastoma tissue microarrays. The biological function was analyzed in vitro by targeted siRNA-mediated knockdown experiments in 3 hepatoblastoma cell lines (HepT1, HepG2, HuH6) und subsequent cell growth assays (MTT) and WNT signaling pathway specific luciferase reporter assays. Gain of chromosome 2q was present in 44.7% of the cases. Amplification of the 2q24.2-24.3 region was detected in 11.8%. In the smallest overlapping amplified region of 5.6 Mbp, we could accurately map 20 protein coding genes, three genes encoding long noncoding RNAs and one gene encoding a micro-RNA. The RBMS1 gene encoding a single-stranded DNA/RNA binding protein showed significant RNA overexpression in 2q24 amplified tumors. This overexpression was validated by immunohistochemical studies at the protein level. RBMS1 knockdown by specific siRNA transfection resulted in a significantly reduced proliferation of hepatoblastoma cells. RBMS1 knockdown also resulted in a marked reduction of the WNT pathway activity in these cells. We identified RBMS1 located within the amplicon on chromosome 2q24 as a potential oncogenic driver in hepatoblastoma. Initial data suggest that RBMS1 may exert this function by interaction with the WNT signaling pathway that is pathologically activated in hepatoblastoma. Citation Format: Martin Rodemann, Verena Dreschmann, Evelyn Dörner, Dietrich von Schweinitz, Christian Vokuhl, Torsten Pietsch. Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2492.

Abstract 2674: Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and > 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values < 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2674.

Physical Regularities for Cellular Precipitation of Co-, Cu-, and Pb-Based Supersaturated Solid Solutions

The decomposition of supersaturated solid solutions through the cellular mechanism is considered in terms of the physical regularities of this phenomenon. The general characteristics of this process are described. The mechanisms of nucleation and subsequent cell growth, as well as kinetic parameters of processes, are partially described. The influences of some external factors on the cellular precipitation process and its stages are characterized. Particularly, the effects of annealing temperature and a third element on the cellular precipitation process are studied.

Recycled PET foaming: Supercritical carbon dioxide assisted extrusion with real-time quality monitoring

Foaming of recycled poly(ethylene terephthalate) (rPET) was performed by supercritical carbon dioxide (sc-CO 2 ) assisted extrusion. The intrinsic viscosity (IV) of rPET was increased from 0.62 dl/g to 0.87 dl/g using an epoxy-functional chain extender, which provided adequate rheological properties for cell stabilization so that an apparent density of less than 0.15 g/cm 3 became achievable. Homogeneous and talc induced heterogeneous crystal and cell nucleation, subsequent cell growth and stabilization processes were examined using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM), respectively. It was found that using talc the crystallization temperature increases which results in smaller cell size distribution. A strong correlation was evinced between the apparent density and the Fourier transform near-infrared (NIR) spectrum of the foamed rPET samples enabling quick and non-destructive characterization. Accordingly, NIR spectroscopy is demonstrated as a suitable method for in-line quality monitoring during extrusion foaming of recycled PET, being especially prone to quality fluctuations.

Abstract PS18-09: Novel chimeric small molecule AC682 potently degrades estrogen receptor with oral anti-tumor efficacy superior to fulvestrant

Abstract Modulation of estrogen activity and/or synthesis has been the mainstay therapeutic strategy for estrogen receptor (ER)-positive breast cancer. Fulvestrant, which provides superior clinical benefit over aromatase inhibitors and selective ER modulators, achieves its potent ER signaling inhibition through unique ER degradation activity beyond direct antagonism. Despite this superiority, the efficacy of fulvestrant appears limited by poor drug exposure through intramuscular injection. Up to 50% of ER baseline levels remained after 6 months of fulvestrant treatment. Therefore, an oral ER degrader with optimal pharmacokinetic profile may lead to better convenience and efficacy than fulvestrant. Here we report preclinical characterization of our novel oral ER degrader development candidate, AC682, a chimeric small molecule containing both ligands of ER and E3 ligase discovered with our ACCU-Degron technology. Through engaging E3 ligase and ER simultaneously, AC682 induced potent ER degradation with a sub-nanomolar half-maximal degradation concentration (DC50) in multiple ER-positive breast cancer cell lines tested, including a tamoxifen-resistant long-term estrogen deprived (LTED) cell line and cell lines expressing clinically-relevant ESR1 variants (Y537S and D538G). Proteasome-dependent ER degradation by AC682 peaked after a few hours of treatment, resulting in diminished expression of ER-regulated genes and subsequent cell growth inhibition. In the in vivo setting, oral dosing of AC682 achieved substantial drug exposure and oral bioavailability, with well tolerance in multiple animal species. In estradiol-dependent MCF7 xenograft tumors, oral daily dosing of AC682 led to dose-dependent tumor growth inhibition/regression and concomitant tumor ER protein reduction more than 90% at study termination. In particular, 3 mg/kg oral daily dosing achieved tumor stasis. When administrated in combination with CDK4/6 inhibitor Palbociclib, AC682 showed clear synergy in both the estradiol-dependent MCF7 model and a tamoxifen-resistant MCF7 model. In a patient-derived xenograft model, which bears ESR1 Y537S mutation and is hormone-independent, AC682 significantly inhibited tumor growth whereas fulvestrant only showed weak efficacy. In conclusion, oral chimeric ER degrader AC682 demonstrated robust ER degradation and anti-tumor efficacy in preclinical models, supporting its continued investigation in the clinic. Citation Format: Wei He, Hui Zhang, Lorena Perkins, Lissette Bouza, Ke Liu, Yimin Qian, Jie Fan. Novel chimeric small molecule AC682 potently degrades estrogen receptor with oral anti-tumor efficacy superior to fulvestrant [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-09.

The Small Molecule BIBR1532 Exerts Potential Anti-cancer Activities in Preclinical Models of Feline Oral Squamous Cell Carcinoma Through Inhibition of Telomerase Activity and Down-Regulation of TERT.

Expression of telomerase reverse transcriptase (TERT) and telomerase activity (TA) is a main feature of cancer, contributing to cell immortalization by causing telomeres dysfunction. BIBR1532 is a potent telomerase inhibitor that showed potential anti-tumor activities in several types of cancer, by triggering replicative senescence and apoptosis. In a previous work, we detected, for the first time, TERT expression and TA in preclinical models of feline oral squamous cell carcinoma (FOSCC); therefore, we aimed at extending our investigation by testing the effects of treatment with BIBR1532, in order to explore the role of telomerase in this tumor and foreshadow the possibility of it being considered as a future therapeutic target. In the present study, treatment of FOSCC cell lines SCCF1, SCCF2, and SCCF3 with BIBR1532 resulted in successful inhibition of TA, with subsequent cell growth stoppage and decrease in cell viability. Molecular data showed that up-regulation of cell cycle inhibitor p21, unbalancing of Bax/Bcl-2 ratio, and down-regulation of survival gene Survivin were mostly involved in the observed cellular events. Moreover, BIBR1532 diminished the expression of TERT and its transcriptional activator cMyc, resulting in the down-regulation of epidermal growth factor receptor (EGFR), phospho-ERK/ERK ratio, and matrix metalloproteinases (MMPs)-1/-2 and−9, likely as a consequence of an impairment of TERT extra-telomeric functions. Taken together, our data suggest that BIBR1532 exerts multiple anti-cancer activities in FOSCC by inhibiting telomerase pathway and interfering with signaling routes involved in cell proliferation, cell survival, and invasion, paving the way for future translational studies aimed at evaluating its possible employment in the treatment of this severe tumor of cats.

Preparation and Characterization of Iron-Doped Tricalcium Silicate-Based Bone Cement as a Bone Repair Material.

Iron is one of the trace elements required by human body, and its deficiency can lead to abnormal bone metabolism. In this study, the effect of iron ions on the properties of tricalcium silicate bone cement (Fe/C3Ss) was investigated. It effectively solved the problems of high pH value and low biological activity of calcium silicate bone cement. The mechanical properties, in vitro mineralization ability and biocompatibility of the materials were systematically characterized. The results indicate that tricalcium silicate bone cement containing 5 mol% iron displayed good self-setting ability, mechanical properties and biodegradation performance in vitro. Compared with pure calcium silicate bone cement (C3Ss), Fe/C3Ss showed lower pH value (8.80) and higher porosity (45%), which was suitable for subsequent cell growth. Immersion test in vitro also confirmed its good ability to induce hydroxyapatite formation. Furthermore, cell culture experiments performed with Fe/C3Ss ion extracts clearly stated that the material had excellent cell proliferation abilities compared to C3Ss and low toxicity. The findings reveal that iron-doped tricalcium silicate bone cement is a promising bioactive material in bone repair applications.

Integrated consolidated bioprocessing for simultaneous production of Omega-3 fatty acids and bioethanol

Lignocellulosic biomass represents a promising feedstock for supplying biofuels and other useful bioproducts in near future. As an example of process innovation, this study describes an integrated microbial fermentation process involving a thermoanaerobe and a marine thraustochytrid, aiming at the co-production of docosahexaenoic acids and bioethanol from rice straw biomass as substrate. Key points of the process are: (a) consolidated bioprocessing of pretreated rice straw biomass to bioethanol (anaerobic fermentation) resulting into 1.8 g L−1 bioethanol and 29.40% solubilization of rice straw biomass; (b) utilization of spent lignocellulose derived sugars in microalgal fermentation (aerobic fermentation) with subsequent promising cell growth (2.77 g L−1), substantial lipids (17.05%) and docosahexaenoic acids production (44.0% of total fatty acids). Other major fatty acids (as total fatty acid %) were palmitic acid (13.95%), stearic acid (5.07%), eicosapentaenoic acid (7.24%), and docosapentaenoic acid (16.12%). This study provides a new route for marine algae utilization of lignocellulosic biomass for co-production of bioethanol and value-added bioproducts.

Retroviral Insertional Mutagenesis in Humans: Evidence for Four Genetic Mechanisms Promoting Expansion of Cell Clones

Integration of new DNA into a cellular chromosome can alter the activity of nearby genes, sometimes affecting subsequent cell growth. A potent form of insertional mutagenesis involves integration of retroviral DNA produced by reverse transcription, a required step in the replication of retroviruses. In recent years retroviral replication has been adapted to allow new gene addition by retroviral vectors. Early in the history of retrovirus research, analysis of insertional mutagenesis in laboratory animals was found at times to result in transformation, leading to the discovery of cellular proto-oncogenes. In-depth analysis of the genetic consequences showed that integration of retroviral DNA could alter the gene activity in a variety of ways. Mechanisms of retroviral insertional mutagenesis in humans are much less well documented. However, recent work from the gene therapy and HIV fields now specify four genetic mechanisms of retroviral insertional mutagenesis in humans: (1) gene activation by integration of an enhancer sequence encoded in a retroviral vector (enhancer insertion), (2) gene activation by promoter insertion, (3) gene inactivation by insertional disruption, and (4) gene activation by mRNA 3' end substitution. In each example, integration in patients was associated with clonal expansion or frank transformation.

ATF6 Regulates Cardiac Hypertrophy by Transcriptional Induction of the mTORC1 Activator, Rheb.

Endoplasmic reticulum (ER) stress dysregulates ER proteostasis, which activates the transcription factor, ATF6 (activating transcription factor 6α), an inducer of genes that enhance protein folding and restore ER proteostasis. Because of increased protein synthesis, it is possible that protein folding and ER proteostasis are challenged during cardiac myocyte growth. However, it is not known whether ATF6 is activated, and if so, what its function is during hypertrophic growth of cardiac myocytes. To examine the activity and function of ATF6 during cardiac hypertrophy. We found that ER stress and ATF6 were activated and ATF6 target genes were induced in mice subjected to an acute model of transverse aortic constriction, or to free-wheel exercise, both of which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO [conditional knockout]) blunted transverse aortic constriction and exercise-induced cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript profiling and chromatin immunoprecipitation identified RHEB (Ras homologue enriched in brain) as an ATF6 target gene in the heart. RHEB is an activator of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a major inducer of protein synthesis and subsequent cell growth. Both transverse aortic constriction and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in wild type mouse hearts but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine- and IGF1 (insulin-like growth factor 1)-mediated RHEB induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, adeno-associated virus 9- RHEB restored cardiac growth to ATF6 cKO mice subjected to transverse aortic constriction. Finally, ATF6 induced RHEB in response to growth factors, but not in response to other activators of ATF6 that do not induce growth, indicating that ATF6 target gene induction is stress specific. Compensatory cardiac hypertrophy activates ER stress and ATF6, which induces RHEB and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.

The Drosophila Blood-Brain Barrier Adapts to Cell Growth by Unfolding of Pre-existing Septate Junctions

The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. Here, we used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. We show that septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish.

Development of the sphagnoid areolation pattern in leaves of Palaeozoic protosphagnalean mosses.

Protosphagnalean mosses constitute the largest group of extinct mosses of still uncertain affinity. Having the general morphology of the Bryopsida, some have leaves with an areolation pattern characteristic of modern Sphagna. This study describes the structure and variation of these patterns in protosphagnalean mosses and provides a comparison with those of modern Sphagna. Preparations of fossil mosses showing preserved leaf cell structure were obtained by dissolving rock, photographed, and the resulting images were transformed to graphical format and analysed with Areoana computer software. The sphagnoid areolation pattern is identical in its basic structure for both modern Sphagnum and Palaeozoic protosphagnalean mosses. However, in the former group the pattern develops through unequal oblique cell divisions, while in the latter the same pattern is a result of equal cell divisions taking place in a specific order with subsequent uneven cell growth. The protosphagnalean pathway leads to considerable variability in leaf structure. Protosphagnalean mosses had a unique ability to switch the development of leaf areolation between a pathway unique to Sphagnum and another one common to all other mosses. This developmental polyvariancy hinders attempts to classify these mosses, as characters previously considered to be of generic significance can be shown to co-occur in one individual leaf. New understanding of the ontogeny has allowed us to re-evaluate the systematic significance of such diagnostic characters in these Palaeozoic plants, showing that their similarity to Sphagnum is less substantial.

O-GlcNAcylation of the Tumor Suppressor FOXO3 Triggers Aberrant Cancer Cell Growth.

Posttranslational modifications of tumor suppressors can induce abnormal cell growth. Here, we identify site-specific O-GlcNAcylation as a critical block of FOXO3 that may abrogate a part of the p53 pathway, resulting in aberrant cancer cell growth. Of seven O-GlcNAcylation sites identified within the FOXO3 transactivation domain, we found that changes in O-GlcNAcylation at Ser284 modulated p21-mediated cancer cell growth. Overexpression of either O-GlcNAcylated FOXO3 (FOX-OV) or a Ser-to-Ala mutant (S284A) in PANC-1 cells indicated that S284 O-GlcNAc acts as a critical block of the FOXO tumor suppressor and induces proliferation in PANC-1 cancer cells by stimulating the MDM2-p53-p21 axis. Furthermore, S284A mutant cells lacking S284 O-GlcNAc and FOX-OV cells exhibited opposing MDM2-p53-p21 axis expression patterns at both the mRNA and protein levels. Thus, our study provides evidence to support a role for S284 O-GlcNAc as a critical block of FOXO3 to induce subsequent cancer cell growth via abrogation of the p53 regulatory circuit.Significance: These findings highlight a posttranslational mechanism for indirect abrogation of the p53 pathway, one that may occur with some frequency in human cancer cells. Cancer Res; 78(5); 1214-24. ©2018 AACR.

GLUT12 promotes prostate cancer cell growth and is regulated by androgens and CaMKK2 signaling.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.