Suppression of urokinase expression and invasion by a soybean Kunitz trypsin inhibitor are mediated through inhibition of Src-dependent signaling pathways.
A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.
Inagaki
Kiyokazu
K
NetForce Co. Ltd., Taiko 3-1-18, Nakamura, Nagoya, Aichi 453-0801, Japan.
Kobayashi
Hiroshi
H
Yoshida
Ryuji
R
Kanada
Yasufumi
Y
Fukuda
Yoichi
Y
Yagyu
Tatsuo
T
Kondo
Toshiharu
T
Kurita
Noriyuki
N
Kitanaka
Takashi
T
Yamada
Yoshihiko
Y
Sakamoto
Yoshiharu
Y
Suzuki
Mika
M
Kanayama
Naohiro
N
Terao
Toshihiko
T
D045744:Cell Line, Tumor; D004794:Enzyme Repression / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology*; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000502:physiology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology*
D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean; D014568:Urokinase-Type Plasminogen Activator; D019061:src-Family Kinases; C508960:TGFB1 protein, human; C494918:AKT1 protein, human