Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.
AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.
Department of Medicine, University of Louisville, Louisville, KY, 40202, USA; Molecular Targets Program of the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA.
E Merit
EM
Reyes-Reyes
Department of Biochemistry, University of Louisville, Louisville, KY, 40202, USA; Molecular Targets Program of the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA.
Francesca R
FR
Šalipur
Molecular Targets Program of the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA.
Mitra
M
Shams
Molecular Targets Program of the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA.
Matthew K
MK
Forsthoefel
Department of Medicine, University of Louisville, Louisville, KY, 40202, USA; Department of Biochemistry, University of Louisville, Louisville, KY, 40202, USA; Molecular Targets Program of the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA. Electronic address: paula.bates@louisville.edu.
Paula J
PJ
Bates
D000818:Animals; D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000502:physiology*; D010873:Pinocytosis / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D000097796:Nucleolin
D052157:Aptamers, Nucleotide; D066246:ErbB Receptors; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein; D000097796:Nucleolin; C513936:AGRO 100
AS1411; Aptamer; Macropinocytosis; Methuosis; Nucleolin; Rac1