Inhibition of c-Myc down-regulation by sustained extracellular signal-regulated kinase activation prevents the antimetabolite methotrexate- and gemcitabine-induced differentiation in non-small-cell lung cancer cells.
Non-small-cell lung cancer (NSCLC) is characterized by severe resistance to chemotherapy. Here, we demonstrate that A549 adenocarcinoma cells permanently differentiate with the antimetabolites methotrexate (MTX) and gemcitabine (GE) when blocking the resistance mechanism that normally counteracts this process. MTX (1-10 microM) and GE (1 microM) induced growth arrest accompanied by sustained extracellular signal-regulated kinase (ERK1/2) phosphorylation and moderate reduction of c-Myc levels after 96 h, whereas only a low percentage of the cells differentiated. Combination with the mitogen-activated protein kinase kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis-(methylthio)butadiene (U0126) reduced MTX- or GE-induced ERK1/2 over-phosphorylation, nearly abolished c-Myc expression, and provoked radical morphological changes in all cells. Besides the appearance of multilamellar bodies and intracellular cytokeratin reorganization, modulation of molecular markers occurred in a manner consistent with differentiation (gelsolin, +300%; surfactant protein A and C, -70%). Similar to U0126, c-Myc inactivation with specific small interfering RNA initiated differentiation only in the presence of MTX, demonstrating that inhibition of the mitogen-activated protein kinase/ERK pathway alone or down-regulation of c-Myc is not sufficient to induce this process. It is noteworthy that withdrawal of antitumoral drugs and U0126 neither reversed differentiation nor reactivated proliferation. Our results reveal that maintenance of a certain threshold of c-Myc expression through sustained ERK1/2 activation represents a molecular mechanism that confers resistance to antimetabolite-induced differentiation in A549 cells, and provide a novel molecular basis for therapeutic strategies based on irreversible differentiation of cancer cells using conventional chemotherapeutic antimetabolites in combination with inhibitors of the MEK/ERK pathway or c-Myc.
Serra
Jordi M
JM
Department of Hematology, Institut Universitari d'Investigacions en Ciències de la Salut, University Hospital Son Dureta, c/Andrea Dòria 55, E-07014 Palma de Mallorca, Spain. jmserra@hsd.es
Gutiérrez
Antonio
A
Alemany
Regina
R
Navarro
María
M
Ros
Teresa
T
Saus
Carlos
C
Ginés
Jordi
J
Sampol
Antonia
A
Amat
Juan Carlos
JC
Serra-Moisés
Lorenzo
L
Martín
Javier
J
Galmés
Antonio
A
Vögler
Oliver
O
Besalduch
Joan
J
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; C116115:MYCBP protein, human; D014157:Transcription Factors; D003841:Deoxycytidine; D048049:Extracellular Signal-Regulated MAP Kinases; D008727:Methotrexate; D000093542:Gemcitabine