17-allylamino-17-demethoxygeldanamycin and MEK1/2 inhibitors kill GI tumor cells via Ca2+-dependent suppression of GRP78/BiP and induction of ceramide and reactive oxygen species.
The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca(2+) and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.
Walker
Teneille
T
Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia, USA.
Mitchell
Clint
C
Park
Margaret A
MA
Yacoub
Adly
A
Rahmani
Mohamed
M
Häussinger
Dieter
D
Reinehr
Roland
R
Voelkel-Johnson
Christina
C
Fisher
Paul B
PB
Grant
Steven
S
Dent
Paul
P
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000008:administration & dosage / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D002518:Ceramides / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004347:Drug Interactions; D000091342:Endoplasmic Reticulum Chaperone BiP; D005770:Gastrointestinal Neoplasms / Q000378:metabolism / Q000473:pathology*; D045325:HCT116 Cells; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D056945:Hep G2 Cells; D006801:Humans; D047029:Lactams, Macrocyclic / Q000008:administration & dosage / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
D016227:Benzoquinones; D002518:Ceramides; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; C112765:tanespimycin; C482128:MAP2K2 protein, human; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D002118:Calcium