Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Oncogene ; 38(28): 5658-5669, 2019 07.
Article in English | MEDLINE | ID: mdl-30996246

ABSTRACT

BET bromodomain inhibitors block prostate cancer cell growth at least in part through c-Myc and androgen receptor (AR) suppression. However, little is known about other transcriptional regulators whose suppression contributes to BET bromodomain inhibitor anti-tumor activity. Moreover, the anti-tumor activity of BET bromodomain inhibition in AR-independent castration-resistant prostate cancers (CRPC), whose frequency is increasing, is also unknown. Herein, we demonstrate that BET bromodomain inhibition blocks growth of a diverse set of CRPC cell models, including those that are AR-independent or in which c-Myc is not suppressed. To identify transcriptional regulators whose suppression accounts for these effects, we treated multiple CRPC cell lines with the BET bromodomain inhibitor JQ1 and then performed RNA-sequencing followed by Master Regulator computational analysis. This approach identified several previously unappreciated transcriptional regulators that are highly expressed in CRPC and whose suppression, via both transcriptional or post-translational mechanisms, contributes to the anti-tumor activity of BET bromodomain inhibitors.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/metabolism , Transcription Factors/antagonists & inhibitors , Animals , Azepines/pharmacology , Benzamides , Cell Cycle Proteins/physiology , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/physiology , Gene Expression Regulation, Neoplastic/physiology , Humans , Male , Mice , Mice, SCID , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Biosynthesis , Transcription Factors/physiology , Transcription, Genetic , Triazoles/pharmacology
2.
Cell Oncol (Dordr) ; 41(4): 395-408, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29679238

ABSTRACT

BACKGROUND: Lung cancer is the leading cause of cancer-related death in both men and women. KRAS mutations occur in ~ 25% of patients with lung cancer, and the presence of these mutations is associated with a poor prognosis. Unfortunately, efforts to directly target KRAS or its associated downstream MAPK or PI3K/AKT/mTOR pathways have seen little or no benefits. Here, I hypothesize that KRAS-mutant tumors do not respond to KRAS pathway therapies due to the co-occurrence of other activated cell survival pathways and/or mechanisms. METHODS AND RESULTS: To identify other potentially activated cell survival pathways in KRAS-mutant tumors, I performed association rule mining on somatic mutations in 725 metastatic lung cancer patient samples. I identified 67 additional genes that were mutated in at least 10% of the samples with KRAS mutations. This gene list was enriched with genes involved in the MAPK, AKT and STAT3 pathways, as well as in cell-cell adhesion, DNA repair, chromatin remodeling and the Wnt/ß-catenin pathway. I also identified 160 overlapping subsets of three or more genes that code for oncogenic or tumor suppressive proteins that were mutated in at least 10% of the KRAS-mutant tumors. CONCLUSIONS: I identified several genes that are co-mutated in primary KRAS-mutant lung cancer samples. I also identified subpopulations of KRAS-mutant lung cancers based on sets of genes that were co-mutated. Pre-clinical models that capture these subsets of KRAS-mutant tumors may enhance our understanding of lung cancer development and, in addition, facilitate the design of personalized treatment strategies for lung cancer patients carrying KRAS mutations.


Subject(s)
Lung Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Female , Humans , Machine Learning , Male , Mutation , Oncogene Protein v-akt/genetics , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
3.
Sci Rep ; 8(1): 5797, 2018 04 11.
Article in English | MEDLINE | ID: mdl-29643381

ABSTRACT

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.


Subject(s)
Connexins/metabolism , Electrophysiological Phenomena/drug effects , Nerve Tissue Proteins/metabolism , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/physiology , Animals , Electroretinography , Evoked Potentials, Visual , Mice, Inbred C57BL , Mice, Knockout , Patch-Clamp Techniques
4.
J Biol Chem ; 291(17): 9133-47, 2016 Apr 22.
Article in English | MEDLINE | ID: mdl-26895961

ABSTRACT

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gß subunit, Gß5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gß5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gß5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.


Subject(s)
Carrier Proteins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Protein Multimerization/physiology , RGS Proteins/metabolism , Animals , Carrier Proteins/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , RGS Proteins/genetics
5.
Biochemistry ; 54(4): 1077-88, 2015 Feb 03.
Article in English | MEDLINE | ID: mdl-25551629

ABSTRACT

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gß5-RGS7, the permanently associated heterodimer of G protein ß-subunit Gß5 and RGS7, a regulator of G protein signaling. Gß5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gß5-RGS7. In addition to the i3 loop, interaction of M3R with Gß5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gß5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gß5-RGS7 requires helix 8 and the central portion of the i3 loop.


Subject(s)
GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/physiology , Receptor, Muscarinic M3/chemistry , Receptor, Muscarinic M3/physiology , Amino Acid Sequence , Animals , Binding Sites/physiology , Cholinergic Agents/pharmacology , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Molecular Sequence Data , Receptor, Muscarinic M3/agonists
6.
Int J Biol Macromol ; 75: 37-43, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25603139

ABSTRACT

This study demonstrates the requirement of Asp-380 and Asp-386 in the ßDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants ßD380A/ßD386A, ßD380Q/ßD386Q, or ßD380R/ßD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of ßD380 and ßD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of ßDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, ßD380 and ßD386, are essential for proper peptide binding and inhibition of ATP synthase.


Subject(s)
ATP Synthetase Complexes/metabolism , Aspartic Acid/metabolism , Escherichia coli/enzymology , Peptides/chemistry , ATP Synthetase Complexes/antagonists & inhibitors , Amino Acid Motifs , Amino Acid Sequence , Cell Membrane/enzymology , Crystallography, X-Ray , Escherichia coli/drug effects , Escherichia coli/growth & development , Glucose/pharmacology , Melitten/chemistry , Melitten/pharmacology , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Protein Binding/drug effects , Structure-Activity Relationship , Succinic Acid/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...