RESUMO
Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 µM, consistent with a Ki of 3.50 µM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.
Assuntos
Lisofosfatidilcolinas/uso terapêutico , Melanoma/tratamento farmacológico , Sulfonas/uso terapêutico , Linhagem Celular Tumoral , Humanos , Lisofosfatidilcolinas/administração & dosagem , Neovascularização Patológica , Sulfonas/administração & dosagemRESUMO
BACKGROUND: A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. RESULTS: In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. CONCLUSIONS: Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteínas RGS/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Biologia Computacional , Docetaxel , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lisofosfolipídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase , Proteínas RGS/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Taxoides/farmacologia , Vincristina/farmacologiaRESUMO
BACKGROUND: Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective. RESULTS: Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor. CONCLUSIONS: Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.
Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Ácidos Fosfatídicos/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Complexos Multienzimáticos/antagonistas & inibidores , Fosfodiesterase I/antagonistas & inibidores , Diester Fosfórico Hidrolases , Pirofosfatases/antagonistas & inibidores , RNA Interferente Pequeno , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The hyperactive FVB/N inbred mouse strain is widely used for transgenic research applications, although rarely for behavioral studies. These mice have visual impairments via retinal degeneration, but are considered highly intelligent and rely largely on olfaction. While investigating diet-induced obesity in autotaxin transgenic FVB/N mice, we observed an increase in the necessity for male, but not female, cage separations. Based on the observations, we hypothesized that feeding FVB/N mice a lean diet increases nocturnal bouts of aggression between male littermates. The diets of adult littermates were switched from normal chow to either ad libitum high-fat (45% fat) or lean (10% fat) matched diets for 27 weeks, whereby the mice reached an average of 43 g versus 35 g, respectively. Then, cage separations due to nocturnal bouts of aggression became mandatory, even though littermates peacefully cohabitated for 10-16 weeks previously. Since the data was of an unusual nature, it required uncommon statistical methods to be engendered to evaluate whether and where significance existed. Therefore, utilizing the randomization and population models, we established a methodology and postulated that either testosterone, the autotaxin transgene or diet alteration was the causal factor. Statistical evaluation demonstrated a significant correlation between cage separations and aggressive behavior associated with the lean-diet-fed mice, not autotaxin. Biochemical data did not appear to explain the behavior. In contrast, energy metabolism highlighted differences between the groups of normally hyperactive mice by diet. This characteristic makes FVB/N male mice unsuitable subjects for long-term studies with lean-diet modifications.
Assuntos
Agressão , Dieta com Restrição de Gorduras/efeitos adversos , Animais , Peso Corporal , Masculino , Camundongos , Camundongos Endogâmicos , IrmãosRESUMO
Since the molecular cloning of the vzg-1/Edg-2/LPA1 gene, studies have attempted to characterize LPA1 receptor functionality into a single categorical role, different from the other Edg-family LPA receptors. The desire to categorize LPA1 function has highlighted its complexity and demonstrated that the LPA1 receptor does not have one absolute function throughout every system. The central nervous system is highly enriched in the LPA1 receptor, suggesting an integral role in neuronal processes. Metastatic and invasive breast cancer also appears to have LPA-mediated LPA1 receptor functions that enhance phenotypes associated with tumorigenesis. LPA1 possesses a number of motifs conserved among G protein-coupled receptors (GPCRs): a DRY-like motif, a PDZ domain, Ser/Thr predicted sites of phosphorylation, a di-leucine motif, double cysteines in the tail and conserved residues that stabilize structure and determine ligand binding. The third intracellular loop of the LPA1 receptor may be the crux of receptor signaling and attenuation with phosphorylation of Thr-236 potentially a key determinant of basal LPA1 signaling. Mutagenesis data supports the notion that Thr-236 regulates this process since mutating Thr-236 to Ala-236 increased basal and LPA-mediated serum response factor (SRF) signaling activity and Lys-236 further increased this basal signaling. Here we describe progress on defining the major functions of the LPA1 receptor, discuss a context dependent dualistic role as both a negative regulator in cancer and a proto-oncogene, outline its structural components at the molecular amino acid level and present mutagenesis data on the third intracellular loop of the receptor.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Progressão da Doença , Humanos , Neoplasias/genética , Neoplasias/patologia , Proto-Oncogene Mas , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de SinaisRESUMO
The revelation that microRNAs (miRNAs) exist within the human genome uncovered an underappreciated mechanism of gene expression. For cells to regulate expression of their genes, miRNA molecules and argonaute proteins bind to mRNAs and interfere with efficient translation of the RNA transcript. Although miRNAs have important roles in normal tissues, miRNAs may adopt aberrant functions in malignant cells depending on their classification as either a tumor suppressor or oncogenic miRNA. Within this review, the current status of miRNA regulation is described in the context of signaling through the lysophosphatidic acid receptors, including the lysophosphatidic acid-producing enzyme, autotaxin. Thus far, research has revealed miRNAs that increase in response to lysophosphatidic acid stimulation, such as miR-21, miR-30c-2-3p, and miR-122. Other miRNAs inhibit the translation of lysophosphatidic acid receptors, such as miR-15b, miR-23a, and miR200c, or proteins that are downstream of lysophosphatidic acid signaling, such as miR-146 and miR-21. With thousands of miRNAs still uncharacterized, it is anticipated that the complex regulation of lysophosphatidic acid signaling by miRNAs will continue to be elucidated. RNA-based therapeutics have entered the clinic with enormous potential in precision medicine. This exciting field is rapidly emerging and it will be fascinating to witness its expansion in scope.
RESUMO
The lysophosphatidic acid receptor-3 (LPAR3) is a G protein-coupled receptor that mediates viability among malignant cells and aggressiveness among certain tumors. The study's objective was to determine the interplay between LPAR3 and miRNAs to impact key cellular signaling pathways. Using SK-Mel-2 and SK-Mel-5 melanoma cells, wild-type and mutated receptors were stably expressed to explore molecular mechanisms. LPAR3 signaling induced miR-122-5p intracellularly and subsequently its inclusion into exosomes. This amplification resulted in less abundant Wnt1, maintenance of GSK3 inactivation and to a lesser extent, partial degradation of ß-catenin. The surge in miR-122-5p and reduction in Wnt1 originated from signaling at the Src homology 3 (SH3) ligand-binding motif within the third intracellular loop of LPAR3, because mutant receptors did not increase miR-122-5p and had a weakened capacity to reduce Wnt1. In addition, a key mediator of melanoma survival signaling, the peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A/PGC1), was involved in miR-122-5p transcription. In conclusion, this study highlights the powerful role miRNAs have in fine-tuning specific G protein-coupled receptor-mediated signaling events by altering the transcription of signaling transduction pathway components. This study also identifies that LPAR3 increases miR-122-5p expression, which occurs mechanistically through the SH3 domain and helps explain why miR-122-5p increases are detected in cancer patient serum. IMPLICATIONS: LPAR3 is partially responsible for the production and secretion of miR-122-5p, found in the serum of a wide variety of patients with cancer.
Assuntos
Melanoma/metabolismo , MicroRNAs/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteína Wnt1/metabolismo , Animais , Sítios de Ligação , Proliferação de Células/fisiologia , Células Hep G2 , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , MicroRNAs/genética , Domínios ProteicosRESUMO
In recent years, small endogenous RNAs have come to the forefront of both basic and translational research. For example, many studies have pointed to the potential role of microRNAs (miRNAs) as disease biomarkers. However, precise quantitative methods for the analysis of miRNAs are still lacking. In this study, we report the first mass spectrometry-based quantitation of miR-451, a circulatory microRNA. Using a highly selective sample preparation method with an average recovery of 83.6% and a novel mobile phase chemistry, we were able to reach an LOQ of 0.5 ng/mL. Because of such high sensitivity, we could detect and quantify the endogenous miR-451 from both human and rat plasma. Considering the increased precision of LC-MS compared to other methods, these results usher in a new era of miRNA biomarker discovery and validation.
Assuntos
Biomarcadores/sangue , Cromatografia Líquida/métodos , MicroRNAs/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
Lysophosphatidic acid (LPA) is a bioactive lipid that promotes cancer cell proliferation and motility through activation of cell surface G protein-coupled receptors. Here, we provide the first evidence that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells, which express endogenous LPA receptors. The LPA effect depends on increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. Inhibition of phosphatidylinositol 3-kinase protected cells from the LPA-induced reduction of p53, which implicates this signaling pathway in the mechanism of LPA-induced loss of p53. LPA partially protected A549 cells from actinomycin D induction of both apoptosis and increased p53 abundance. Expression of LPA(1), LPA(2), and LPA(3) receptors in HepG2 hepatoma cells, which normally do not respond to LPA, also decreased p53 expression and p53-dependent transcription. In contrast, neither inactive LPA(1) (R124A) nor another G(i)-coupled receptor, the M(2) muscarinic acetylcholine receptor, reduced p53-dependent transcription in HepG2 cells. These results identify p53 as a target of LPA action and provide a new dimension for understanding how LPA stimulates cancer cell division, protects against apoptosis, and thereby promotes tumor progression.
Assuntos
Lisofosfolipídeos/toxicidade , Mitógenos/toxicidade , Neoplasias/patologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Apoptose/genética , Carcinoma , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Dano ao DNA , Humanos , Neoplasias Pulmonares , Neoplasias/genética , Neoplasias/metabolismo , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/metabolismoRESUMO
Upregulated expression of autotaxin, a secreted phospholipase and phosphodiesterase enzyme, appears in malignant disease. The identification of a circulating miRNA signature should distinguish autotaxin-mediated disease and also elucidate unknown molecular mechanisms that rationalize its malignant potential. Using female transgenic 'AT-ATX' mice, whereby human wild-type autotaxin is expressed in liver under the control of the alpha-1 antitrypsin promoter, transgenic animals express augmented autotaxin in circulation and a percentage develop tumors. Serum collected at necropsy had circulating miRNAs analyzed for statistical significance. The ensuing autotaxin-mediated miRNome differentiated between groups: healthy FVB/N mice versus AT-ATX mice with and without tumors. Intriguingly, miR-489-3p was sharply increased in AT-ATX tumor-bearing mice. Tissue analysis showed a correlation between miR-489-3p expression in tumors and surrounding milieu with autotaxin concentration in circulation. Sequence alignment suggested miR-489-3p targets MEK1, which was confirmed through in vitro studies. Exogenously added miR-489-3p, which decreases MEK1 in normal cells, dramatically increased MEK1 expression in cells stably expressing autotaxin. Taken together, this suggests that autotaxin overrides the normal regulatory function of miR-489-3p to inhibit MEK1 via coordinately increased miR-489-3p appearing in serum.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 1/metabolismo , MicroRNAs/genética , Neoplasias Ovarianas/patologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Biomarcadores Tumorais/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences. Graphical Abstract á .
Assuntos
DNA/química , Oligonucleotídeos/química , Espectrometria de Massas por Ionização por Electrospray , Sequência de Bases , Interações Hidrofóbicas e Hidrofílicas , Indicadores e Reagentes , Íons/química , Prótons , Solubilidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Água/químicaRESUMO
Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase. Graphical Abstract á .
Assuntos
Cromatografia Líquida/métodos , Oligonucleotídeos/química , Propanóis/química , Espectrometria de Massas por Ionização por Electrospray/métodos , DNA/química , Halogenação , Interações Hidrofóbicas e Hidrofílicas , Indicadores e Reagentes , Metilação , RNA/químicaRESUMO
More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Ovário/efeitos dos fármacos , Proteínas RGS/genética , Animais , Antineoplásicos/uso terapêutico , Epigênese Genética , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Proteínas RGS/análise , Proteínas RGS/metabolismoRESUMO
The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.
Assuntos
Apoptose , Proliferação de Células , Melanoma/metabolismo , Melanoma/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Western Blotting , Cálcio/metabolismo , Imunofluorescência , Humanos , Melanoma/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Receptores de Ácidos Lisofosfatídicos/genética , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.
Assuntos
Fator 3 Ativador da Transcrição/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , MicroRNAs/genética , Transcrição Gênica/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Fator 3 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Immunoblotting , Microscopia de Fluorescência , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The regulator of G protein signaling 10 (RGS10) protein is a GTPase activating protein that accelerates the hydrolysis of GTP and therefore canonically inactivates G proteins, ultimately terminating signaling. Rheb is a small GTPase protein that shuttles between its GDP- and GTP-bound forms to activate mTOR. Since RGS10 suppression augments ovarian cancer cell viability, we sought to elucidate the molecular mechanism. Following RGS10 suppression in serum-free conditions, phosphorylation of mTOR, the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), p70S6K and S6 Ribosomal Protein appear. Furthermore, suppressing RGS10 increases activated Rheb, suggesting RGS10 antagonizes mTOR signaling via the small G-protein. The effects of RGS10 suppression are enhanced after stimulating cells with the growth factor, lysophosphatidic acid, and reduced with mTOR inhibitors, temsirolimus and INK-128. Suppression of RGS10 leads to an increase in cell proliferation, even in the presence of etoposide. In summary, the RGS10 suppression increases Rheb-GTP and mTOR signaling in ovarian cancer cells. Our results suggest that RGS10 could serve in a novel, and previously unknown, role by accelerating the hydrolysis of GTP from Rheb in ovarian cancer cells.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas RGS/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Neoplasias Ovarianas/patologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas RGS/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de SinaisRESUMO
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Assuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteína Quinase Tipo II Dependente de AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo I Dependente de AMP Cíclico/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteínas Quinases/química , Subunidades Proteicas/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proteína Quinase Tipo I Dependente de AMP Cíclico/química , Proteína Quinase Tipo I Dependente de AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico/química , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosforilação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Epigenetics is essentially a phenotypical change in gene expression without any alteration of the DNA sequence; the emergence of epigenetics in cancer research and mainstream oncology is fueling new hope. However, it is not yet known whether this knowledge will translate to improved clinical management of ovarian cancer. In this malignancy, women are still undergoing chemotherapy similar to what was approved in 1978, which to this day represents one of the biggest breakthroughs for treating ovarian cancer. Although liquid tumors are benefiting from epigenetically related therapies, solid tumors like ovarian cancer are not (yet?). Herein, we will review the science of molecular epigenetics, especially DNA methylation, histone modifications and microRNA, but also include transcription factors since they, too, are important in ovarian cancer. Pre-clinical and clinical research on the role of epigenetic modifications is also summarized. Unfortunately, ovarian cancer remains an idiopathic disease, for the most part, and there are many areas of patient management, which could benefit from improved technology. This review will also highlight the evidence suggesting that epigenetics may have pre-clinical utility in pharmacology and clinical applications for prognosis and diagnosis. Finally, drugs currently in clinical trials (i.e., histone deacetylase inhibitors) are discussed along with the promise for epigenetics in the exploitation of chemoresistance. Whether epigenetics will ultimately be the answer to better management in ovarian cancer is currently unknown; but we hope so in the future.
RESUMO
RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.