Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.
OBJECTIVE
Notch, a type 1 transmembrane protein, plays a key role in the development of many tissues and organ types. Aberrant Notch signaling, found in a wide variety of human cancers, contributes to tumor development. Because Notch1 was found to be overexpressed in prostate cancer (PCa) cells and human PCa tissue, we therefore tested our hypothesis that overexpression of Notch1 in PCa promotes tumor invasion.


METHODS
Notch1 expression was evaluated in human PCa cells and human PCa tissues. PCa cells were transiently transfected with Notch1-specific small interfering RNAs in concentrations ranging from 30 to 120 nmol/L and subsequently evaluated for effects on invasion and expression analysis for molecules involved in invasion.


RESULTS
Small interfering RNA-mediated knockdown of Notch1 in PC3 and 22Rnu1 PCa cells dramatically decreased their invasion. Focused cDNA array revealed that Notch1 knockdown resulted in significant reduction in the expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) gene transcripts. These data were further verified by reverse transcription-PCR, real-time reverse transcription-PCR, and immunoblot analysis. Knockdown of Notch1 was also observed to significantly reduce the mRNA expression and protein levels of uPA and its receptor uPAR. A significant reduction in MMP9 expression in Notch1 knockdown cells suggested a role for Notch1 in augmenting MMP9 transcription.


CONCLUSIONS
Our data show the involvement of Notch1 in human PCa invasion and that silencing of Notch1 inhibits invasion of human PCa cells by inhibiting the expression of MMP9 and uPA. Thus, targeting of Notch1 could be an effective therapeutic approach against PCa.
Bin Hafeez
Bilal
B
Department of Dermatology, University of Wisconsin, 1300 University Avenue, Medical Science Center, B-25, Madison, WI 53706, USA.
Adhami
Vaqar Mustafa
VM
Asim
Mohammad
M
Siddiqui
Imtiaz A
IA
Bhat
Kumar M
KM
Zhong
Weixiong
W
Saleem
Mohammad
M
Din
Maria
M
Setaluri
Vijayasaradhi
V
Mukhtar
Hasan
H
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D061965:Matrix Metalloproteinase Inhibitors; D009361:Neoplasm Invasiveness; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051881:Receptor, Notch1 / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016896:Treatment Outcome; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D061965:Matrix Metalloproteinase Inhibitors; C497761:NOTCH1 protein, human; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D014568:Urokinase-Type Plasminogen Activator