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1.
Cancer Res ; 80(17): 3719-3731, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32605999

ABSTRACT

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer-disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF-RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. SIGNIFICANCE: These findings show that oncogenic KRASQ61H forms a cooperative RAS-RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , MAP Kinase Signaling System/physiology , Proto-Oncogene Proteins p21(ras)/genetics , raf Kinases/genetics , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Female , HEK293 Cells , Heterografts , Humans , Lung Neoplasms/metabolism , Mice , Mutation , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism
2.
Drug Des Devel Ther ; 9: 1511-54, 2015.
Article in English | MEDLINE | ID: mdl-25834399

ABSTRACT

Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways.


Subject(s)
Apoptosis/drug effects , Naphthoquinones/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Sirtuin 1/metabolism , TOR Serine-Threonine Kinases/metabolism , Autophagy/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Male , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Naphthoquinones/chemistry , Prostatic Neoplasms/pathology , Structure-Activity Relationship , Tumor Cells, Cultured
3.
Drug Metab Lett ; 2(3): 223-30, 2008 Aug.
Article in English | MEDLINE | ID: mdl-19356097

ABSTRACT

The extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we have investigated the role of P-glycoprotein (P-gp) in the transport of tanshinone I (TSI), a major active constituent of S. miltiorrhiza. The TSI transport across Caco-2 monolayers was pH-, energy-, and temperature-dependent, but not sodium-dependent. TSI exhibited a polarized transport in Caco-2 monolayers which was attenuated by P-gp inhibitors. The permeability (P(app)) values of TSI in the basolateral to apical direction were significantly higher in MDCK-II cells over-expressing MDR1, as compared to the wild-type control cells. Furthermore, TSI significantly inhibited the transport of digoxin in Caco-2 cells with an IC(50) value of 0.53 +/- 0.09 microM. TSI also moderately stimulated P-gp ATPase activity with K(m) and V(max) values of 31.70 +/- 7.09 microM and 57.71 +/- 5.26 nmol/min/mg protein, respectively. Our findings indicate that TSI is a substrate and inhibitor of P-gp, which has important clinical and toxicological implications.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Phenanthrenes/pharmacokinetics , Salvia miltiorrhiza/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects , Abietanes , Adenosine Triphosphatases/metabolism , Animals , Biological Transport , Caco-2 Cells , Cell Line , Digoxin/pharmacokinetics , Dogs , Humans , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Multidrug Resistance-Associated Proteins/genetics , Phenanthrenes/administration & dosage , Phenanthrenes/isolation & purification , Plant Roots , Temperature
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