Suppression of human MDA-MB-435S tumor by U6 promoter-driven short hairpin RNAs targeting focal adhesion kinase.
OBJECTIVE
Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cancer cell survival, proliferation, and in various steps in the metastatic cascade. In the present study, we took advantage of a cationic liposome as gene carrier and targeted FAK function through both in vitro and in vivo approaches.


METHODS
We utilized a plasmid-encoded hairpin RNA targeting the human FAK mRNA (pGensil2-shRNA/FAK), as a means to inhibit FAK expression for evaluating its anti-tumor effect in vitro and in vivo. Human MDA-MB-435S breast cancer cells were transfected with pGensil2-shRNA/FAK and examined for apoptosis by propidium iodide staining, DNA ladder, and flow cytometric analysis. For in vivo study, subcutaneous breast carcinomatosis models in nude mice were established to evaluate the therapeutic potential of pGensil2-shRNA/FAK. Assessments of proliferation (Ki-67), apoptosis (TUNEL) and angiogenesis (CD31) were done using immunohistochemical analysis.


RESULTS
Transcripts expressed from plasmid both in vitro and in vivo were identified by northern blot analysis. pGensil2-shRNA/FAK effectively down-regulated the expression of FAK as demonstrated in vitro by real time RT-PCR and western blot analysis, whereas by real time RT-PCR and IHC staining of MDA-MB-435S tumors growing subcutaneously. Breast cancer cells lacking FAK expression undergo apoptosis in vitro. Systemic delivery of cationic liposome-complexed plasmids targeting FAK, resulted in the diminishment of subcutaneous tumor growth beyond the effects observed with liposomes carrying a non-specific shRNA. This diminishment in growth was associated with elevated levels of apoptosis (TUNEL staining), decreased cell proliferation (Ki-67 staining) and diminished endothelial cell density (CD31 staining).


CONCLUSIONS
These results indicate that the systemic delivery of plasmid DNA targeting FAK function using cationic liposome as a gene carrier, represents a promising avenue for breast cancer therapy.
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
An-liang
AL
Huang
Yang
Y
Wan
Dian-ying
DY
Liao
Huo-zhen
HZ
Hu
Lin
L
Wei
Xian-huo
XH
Wang
Yan-jun
YJ
Wen
Jiong
J
Li
Li-juan
LJ
Chen
Bing
B
Kan
Ping
P
Chen
Yong-sheng
YS
Wang
Xiang
X
Chen
Xia
X
Zhao
Hong-xin
HX
Deng
Yu-quan
YQ
Wei
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics*; D044584:Carcinoma, Ductal / Q000235:genetics; D045744:Cell Line, Tumor; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000235:genetics*; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human