title: Differential regulation of autophagy and cell viability by ceramide species.
abstract: The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.
authors: a Department of Biochemistry and Molecular Biology; Virginia Commonwealth University ; Richmond , VA , USA.
authors: Nichola
authors: N
authors: Cruickshanks
authors: Jane L
authors: JL
authors: Roberts
authors: M Danielle
authors: MD
authors: Bareford
authors: Mehrad
authors: M
authors: Tavallai
authors: Andrew
authors: A
authors: Poklepovic
authors: Laurence
authors: L
authors: Booth
authors: Sarah
authors: S
authors: Spiegel
authors: Paul
authors: P
authors: Dent
mesh_terms: D001343:Autophagy; D045744:Cell Line, Tumor; D002470:Cell Survival; D002518:Ceramides; D000068876:Fingolimod Hydrochloride / Q000276:immunology*; D006801:Humans; D000068437:Pemetrexed / Q000276:immunology*; D015398:Signal Transduction
chemical_list: D002518:Ceramides; D000068437:Pemetrexed; D000068876:Fingolimod Hydrochloride
keywords: Ad, adenovirus; Alimta; CMV, empty vector plasmid or virus; CerS, ceramide synthase; CerS2; CerS6; ER, endoplasmic reticulum; ERK, extracellular regulated kinase; FTY720; Gilenya; IP, immunoprecipitation; LASS, longevity assurance gene; MAPK, mitogen activated protein kinase; MEK, mitogen activated extracellular regulated kinase; PI3K, phosphatidyl inositol 3 kinase; PTEN, phosphatase and tensin homolog on chromosome 10; PTX, pemetrexed; Pemetrexed; ROS, reactive oxygen species; S1P; SCR, scrambled; VEH, vehicle.; autophagy; ca, constitutively active; ceramide; dn, dominant negative; mTOR, mammalian target of rapamycin; si, small interfering