Target For Inhibition Of Growth Research Articles

Abstract P005: GRK4 Inhibitors Suppress Breast Cancer Cell Growth And Potentiate The Inhibitory Effect Of Dopaminergic D 1 Receptor Agonist SKF38393

The renal dopaminergic D 1 receptor (D 1 R) regulates sodium excretion which is terminated by phosphorylation by G protein-coupled receptor kinases 4 (GRK4). GRK4 gene variants are associated with increased GRK4 activity and reduced sodium excretion resulting in hypertension. Breast cancer incidence is higher in hypertensive women. We found that GRK4 is a potential molecule linking these two diseases. We hypothesized that GRK4 inhibitors would be beneficial to patients with hypertension and breast cancer. Three potential GRK4 inhibitors (compounds A, B, and C) were tested for their effect on the growth of breast cancer cells MDA-MB-468, MCF-7, and benign mammary epithelial cells MCF-10A controls. These cell lines had high, low, and no GRK4 expression respectively. Growth of MDA-MB-468 and MCF-7 cells was effectively inhibited by compound C with IC 50 16.8±1.9 nM (n=2). MCF-10A cells were relatively resistant to compound C with IC 50 49.3±4.0 nM (n=3) that is significantly higher than the cancer cells (p<0.001). Compound B was the least effective inhibitor in all three cell lines (IC 50 was 1.5-2.3 μM). Growth inhibition of compound A was similar in MCF-7 and MCF-10A cells but less effective in MDA-MB-468 cells indicating GRK4 inhibition may not be the only target for growth inhibition of this compound. It has been reported that D 1 R agonists inhibit growth of breast cancer cells. We hypothesized that blockade of GRK4 would increase sensitivity of breast cancer cells to the inhibitory effect of a D 1 R agonist. In MDA-MB-468 cells, SKF38393 (SKF) at 20 μM caused a 36% reduction in cell number (from 276.7±0.47E4 to 177.7±4.33E4). Compound C alone reduced cell number by 37% (172.4±0.04E4, 5 nM) and 51% (136.3±7.87E4, 10 nM) respectively. Combination treatment induced more reduction in cell number, 63% (100.4±5.54E4, 5 nM) and 84% (44.1±12.7E4, 10 nM). Similarly, Compound A also enhanced the inhibitory effect of SKF. A left-shift of the SKF dose-response curve in GKR4 knock-down MDA-MB-468 cells confirmed that inhibition of GRK4 increases sensitivity of breast cancer cells to SKF. Our preliminary results suggest that targeting GRK4 with compound C and a dopaminergic agonist could be a novel strategy for breast cancer therapy especially for the patients with hypertension.

Abstract 1766: Targeting epigenetic regulation in clear cell renal cell carcinoma reveals PRMT1 as a novel target

Abstract Sporadic Renal Cell Carcinoma (RCC) is dominated by the clear cell subtype (ccRCC) and overwhelmingly associated with a biallelic inactivation of the von Hippel-Lindau (VHL) gene, leading to constitutive activation of the hypoxia response and deleterious alterations to gene expression, metabolism and growth. However, VHL inactivation alone is insufficient to cause cancer. Other key genetic players identified through patient cohort sequencing include frequent inactivating mutations in epigenetic regulatory enzymes. The high frequency of these alterations in ccRCC implicate epigenetic vulnerabilities that may be exploited to develop new therapies.Accordingly, our lab has completed a proliferative screen in patient derived ccRCC models of the Structural Genomics Consortium's (SGC) epiprobe library, a panel of high-quality, small molecule inhibitors directed against an array of epigenetic targets. A particularly favorable inhibition profile was noted for MS023, a probe with potent activity against the type I protein arginine methytransferase family (PRMT1, 3, 4, 6 and 8). PRMTs transfer methyl groups to both nuclear histones and cytoplasmic targets, influencing gene expression, cell signaling, growth and viability. Specific PRMT3, 4 and 6 inhibitors failed to inhibit ccRCC cell growth in our screen, and PRMT8 is not expressed in this cell type, thus PRMT1 is the primary target for growth inhibition of ccRCC by MS023. Upon CRISPR-mediated knockout of PRMT1 followed by a growth competition assay of knock-out vs control cells, all PRMT1-knockout cells dropped out of culture within 4 passages. In ccRCC cell lines with tetracycline-inducible PRMT1 directed shRNAs, cell proliferation was inhibited upon induction with doxycycline both in vitro and in vivo. Additionally, PRMT1 over expression vectors introduced in our ccRCC cell lines, successfully rescued the proliferative phenotype of these cells in the presence of MS023 treatment. Finally, treatment of mice bearing ccRCC xenografts with MS023 led to significant inhibition of tumor growth in vivo. Transcriptomic profiling of our ccRCC models in the presence of MS023 vs control has been performed via RNA Seq and results suggest that this probe is acting as a potent regulator of the cell cycle. We are mapping changes in the H4R3me2a histone mark with MS023 treatment to complement our transcriptomic data and identify direct genetic targets regulated by PRMT1.Evidence continues to mount that dysregulation of PRMT1 is implicated in cancer biology, but to our knowledge, no investigations have been performed in the context of ccRCC. As our nascent understanding of the regulation, function and clinical relevance of arginine methylation continues to expand, this project represents an exciting opportunity to contribute to this body of knowledge, while describing novel therapeutic approaches to ccRCC with the potential for rapid clinical translation. Citation Format: Joseph Paul Walton, Anthony Apostoli, Jalna Meens, Julia Dmytryshyn, Cheryl Arrowsmith, Laurie AIlles. Targeting epigenetic regulation in clear cell renal cell carcinoma reveals PRMT1 as a novel target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1766.

KCa3.1 as an Effective Target for Inhibition of Growth and Progression of Intrahepatic Cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a high malignant tumor arising from the bile ducts in the liver with a poor prognosis. As current molecular targeted therapies and systemic chemotherapies had limited success in ICC, novel therapeutic targets are needed. In this study, we attempted to investigate the expression and the role of the intermediate conductance calcium-activated potassium channel (KCa3.1) in ICC.Methods: The expression levels of KCa3.1 channel were measured in 81 resected ICC tumor specimens and the clinicopathological significance of these levels were determined. KCa3.1 channel inhibitor and siRNA were used to study the role of KCa3.1 in proliferation, migration, and invasion of ICC cell lines. The effect of KCa3.1 channel blockade on tumor growth in vivo was also studied using xenograft model in nude mice.Results: The protein expression of KCa3.1 channel was upregulated in ICC tissues and was correlated with age, lymph node metastasis and TNM stage. And high KCa3.1 expression indicated a worse prognosis in ICC patients. Blocking KCa3.1 channel with a specific inhibitor TRAM-34 reduced the proliferation and invasion of ICC cells. Knockdown of KCa3.1 could achieve the same effects through decreasing NF-κB activation. Further in vivo studies demonstrated that KCa3.1 channel blockade suppressed ICC tumor growth.Conclusions: Our observations suggested KCa3.1 might be a promising novel therapeutic target in intrahepatic cholangiocarcinoma.

A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BackgroundIn the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed.MethodsWe present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen.ResultsUsing the growth inhibition assay, we find a reduction in BCG CFU of 0.3–0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay.ConclusionsWe believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1751-4) contains supplementary material, which is available to authorized users.

Mutant screening for oncogenes of Ewing's sarcoma using yeast.

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.

Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells.

Golgi alpha-mannosidase II, a key enzyme in N-glycan processing, is a target in the development of anti- cancer therapies. The crystal structure of Drosophila Golgi alpha-mannosidase II in the absence and presence of the anti-cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan(5)GlcNAc(2) substrate and the consecutive hydrolysis of the alpha1,6- and alpha1,3-linked mannose residues. The enzyme-inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of alpha-mannosidase II.

Protein kinase C and adenylate cyclase as targets for growth inhibition of human gastric cancer cells.

In the human gastric adenocarcinoma cell line AGS the effects of the protein-kinase-C-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), the protein kinase C inhibitor staurosporine, the adenylate-cyclase activating agent forskolin, and the permeable dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) on the proliferation were assessed. Cell counting followed 5 days of incubation. Prolonged activation of protein kinase C by TPA, inhibition of protein kinase C by staurosporine, activation of adenylate cyclase by forskolin or a direct increase of the intracellular cAMP level all result in a dose-dependent growth inhibition of AGS gastric tumour cells. Half-maximal inhibition was achieved at 100 pM for TPA, 1 nM for staurosporine, 20 microM for forskolin, and 600 microM for Bt2cAMP. It is concluded that protein kinase C and adenylate cyclase play a fundamental role in the growth of AGS gastric cancer cells. Interference with these enzymes involved in the signal transduction of growth regulation in tumour cells may represent a target in the development of new antiproliferative principles.

Polyamine metabolism as a target for growth inhibition of Acanthamoeba culbertsoni.

Alpha-difluoromethylornithine had no inhibitory effect on the growth of Acanthamoeba culbertsoni, while methylglyoxal bis(guanyl-hydrazone) and sinefungin completely blocked amebic growth at 2.5 mM and 5 micrograms/ml concentrations, respectively. Cyclohexylamine caused only partial inhibition of amebic growth at 50-200 microM concentrations. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase of A. culbertsoni indicating this enzyme to be the target for its antiamebic action. This enzyme was, however, not inhibited by sinefungin, suggesting an alternative target for growth inhibition by this compound.

Bacteriostatic and bactericidal activities of beta-lactams against Streptococcus (Enterococcus) faecium are associated with saturation of different penicillin-binding proteins

The MICs and MBCs of benzylpenicillin, ampicillin, cefotaxime, and methicillin were evaluated against a Streptococcus (Enterococcus) faecium wild-type strain and against three mutants hyperproducing PBP 5 in cells incubated at both optimal and suboptimal temperatures. In the wild-type strain grown at optimal temperature, the MBCs of all beta-lactams were significantly greater than the MICs (bacteriostatic effect). As opposed to this, in the same cells grown at suboptimal temperature and in the mutants hyperproducing PBP 5 at all temperatures, the MICs of all antibiotics coincided with the MBCs (bactericidal effect). Under all conditions in which the MIC and MBC were the same, with all antibiotics, growth inhibition occurred only at the minimal concentration saturating all penicillin-binding proteins (PBPs) (or at higher concentrations). On the contrary, under conditions in which the MIC was lower than the MBC, only some of the PBPs were saturated (or bound) at both the MIC and the MBC, PBP 5 in no case being either saturated or bound. Under all conditions in which saturation of all PBPs was needed for growth inhibition, cells died at all antibiotic MBCs with kinetics which were much faster than those with which they died at the MBCs under conditions in which not all PBPs were saturated (or bound). In addition, under the former conditions, antibiotic concentrations above the MBCs did not significantly accelerate cell death kinetics, while under the latter conditions there was an acceleration in kinetics with increasing antibiotic concentrations up to full saturation of PBPs. It is suggested that the killing that occurs when all PBPs are saturated is a direct consequence of inactivation of PBP functions, while killing occurring when only some of them are saturated or bound is also (or mainly) an indirect consequence of inability of cells to grow and that, in S. faecium, the targets for growth inhibition and cell killing reside in different PBPs: for the latter effect, inactivation of one (or more) of the high-molecular-weight PBPs is sufficient, whereas in the former case inactivation of PBP 5 is necessary (after saturation of all other PBPs).