title: Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer.
abstract: BACKGROUND
  Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.
  
  
  RESULTS
  In NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.
  
  
  CONCLUSIONS
  Overall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.
authors.affiliation: Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy. delbufalo@ifo.it.
authors.forename: Donatella
authors.initials: D
authors.lastname: Del Bufalo
authors.forename: Marianna
authors.initials: M
authors.lastname: Desideri
authors.forename: Teresa
authors.initials: T
authors.lastname: De Luca
authors.forename: Marta
authors.initials: M
authors.lastname: Di Martile
authors.forename: Chiara
authors.initials: C
authors.lastname: Gabellini
authors.forename: Valentina
authors.initials: V
authors.lastname: Monica
authors.forename: Simone
authors.initials: S
authors.lastname: Busso
authors.forename: Adriana
authors.initials: A
authors.lastname: Eramo
authors.forename: Ruggero
authors.initials: R
authors.lastname: De Maria
authors.forename: Michele
authors.initials: M
authors.lastname: Milella
authors.forename: Daniela
authors.initials: D
authors.lastname: Trisciuoglio
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D005260:Female; D020868:Gene Silencing / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology*; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000068437:Pemetrexed; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D005971:Glutamates; D056572:Histone Deacetylase Inhibitors; D012333:RNA, Messenger; D000068437:Pemetrexed; D006147:Guanine; D013940:Thymidylate Synthase