The direct effect of focal adhesion kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis.
BACKGROUND
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.


METHODS
To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.


RESULTS
Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.


CONCLUSIONS
Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal specific expression of genes affected by silencing of FAK.
Golubovskaya
Vita M
VM
Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA. Vita.Golubovskaya@Roswellpark.org
Zheng
Min
M
Zhang
Li
L
Li
Jian-Liang
JL
Cance
William G
WG
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D004318:Doxycycline / Q000494:pharmacology; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000235:genetics* / Q000378:metabolism*; D015894:Genome, Human; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D020411:Oligonucleotide Array Sequence Analysis; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; D051417:Focal Adhesion Kinase 1; D004318:Doxycycline