The key findings from the present study are that targeted therapy with FAK siRNA-DOPC in combination with chemotherapy significantly reduces tumor growth in both chemotherapy-sensitive and chemotherapy-resistant models. These effects are likely both direct and indirect (by inducing apoptosis in tumor-associated endothelial cells) at least in part due to reduction of VEGF. Given the clinical relationship between FAK and ovarian cancer prognosis (13
), these findings implicate FAK as an attractive therapeutic target in ovarian cancer.
FAK is a nonreceptor kinase that is a critical mediator of signaling events between cells and their extracellular matrix (35
). It is tyrosine phosphorylated upon integrin binding or ligand binding by growth factor receptors (37
). FAK activation at focal adhesion sites leads to cytoskeletal reorganization, cellular adhesion, and survival, and it is known to play a role in cell migration and invasion (13
). Several reports have linked FAK expression with cancer progression. FAK overexpression has been reported in many malignancies, including ovarian, colon, breast, and head and neck cancers (17
). Specifically, in ovarian cancer, FAK is overexpressed in ~68% of tumors and is associated with high-risk clinical features and poor survival (13
). Although the mechanisms behind FAK overexpression are not fully known, we have recently found that FAK gene amplification is present in ovarian cancers (15
). We have also shown previously that FAK is a substrate for caspase-3 and is degraded during treatment with docetaxel in sensitive cells (17
). Prior in vitro
studies prompted us to examine the therapeutic efficacy of FAK silencing in vivo
, and to the best of our knowledge, our study is the first to use a clinically relevant delivery system for FAK siRNA in an orthotopic model of ovarian cancer. In the current study, therapy was started with low-volume ovarian carcinoma. Although we have shown siRNA delivery into larger tumors previously (23
), whether FAK inhibition has therapeutic efficacy with bulkier tumors remains to be determined.
Although FAK silencing seems to have direct antitumor activity, there is growing evidence that the tumor microenvironment may also be affected. Sheta et al. showed that cell contact-mediated induction of VEGF transcription occurs via FAK (39
). In an elegant study, Mitra et al. showed that 4T1 breast cancer tumors with inhibited FAK activity were substantially smaller compared with controls and exhibited less VEGF staining (30
). Furthermore, cells with mutant FAK had impaired VEGF production and reduced tumor growth in vivo
). In our experiments, FAK silencing was associated with lower levels of VEGF and matrix metalloproteinase-9 and increased apoptosis of tumor-associated endothelial cells, suggesting an antivascular effect.
Because of the pivotal role of FAK in many processes associated with cancer progression, interfering with its function may represent novel therapeutic opportunities. Investigators are developing various strategies for targeting FAK. Strategies aimed at inhibiting FAK kinase activity are being devised (40
). Small-molecule inhibitors that act as competitors for ATP binding at the catalytic site are also being developed. We and others are using siRNA for down-regulating FAK using approaches that may hold promise for clinical use. We have shown previously the feasibility of delivering gene-specific siRNA using DOPC liposomes in orthotopic ovarian cancer models (23
). This approach is more efficient for in vivo
siRNA delivery than either naked siRNA or cationic liposomes. In the current study, we used specific FAK siRNA in DOPC liposomes for systemic delivery, which resulted in efficient FAK down-regulation and therapeutic efficacy. The importance of this work is that the packaging of siRNA into liposomes is rapidly transferable to a clinical setting for cancer therapeutics. It is possible that this delivery method may also be useful for noncancerous diseases amenable to siRNA therapy (41
). This technique is not tissue specific, but further modifications of the liposome may allow tumor-selective delivery (44
Several other approaches for delivering siRNA in vivo
have been attempted. Duxbury et al. showed that systemic delivery of naked siRNA targeting FAK (46
) or EphA2 (47
) down-regulated protein expression and slowed the growth of a single s.c. injected malignant pancreatic cell line. Sorensen et al. showed the reduction of tumor necrosis factor-α expression in liver and spleen by delivering siRNA packaged in cationic liposomes (48
). Others have used rapid injection of a high volume of material (i.e., 2 mL into a mouse with an estimated 4 mL total blood volume) hydrodynamically forcing siRNA into the liver (42
). However, such techniques are likely not practical in a clinical setting.
We investigated FAK silencing using a neutral liposome delivery method for several reasons. Liposomes are already being clinically used for chemotherapy and other delivery systems. Liposomes are a form of nanoparticles that function as carriers and act as a slow-release depot for the drug in the diseased tissue. Optimal liposome size depends on the tumor target. In tumor tissue, the vasculature is discontinuous, and pore sizes vary from 100 to 780 nm (49
). By comparison, pore size in normal vascular endothelium is <2 nm in most tissues and 6 nm in postcapillary venules. Most liposomes are 65 to 125 nm in diameter. Negatively charged liposomes were believed to be more rapidly removed from circulation than neutral or positively charged liposomes; however, recent studies have indicated that the type of negatively charged lipid affects the rate of liposome uptake by the reticuloendothelial system. For example, liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) are cleared more rapidly than neutral liposomes. Cationic liposomes are not ideal delivery vehicles for tumor cells because surface interactions with the tumor cells create an electrostatically derived binding site barrier effect, inhibiting further association of the delivery systems with tumor spheroids (50
). By comparison, neutral liposomes seem to have better intratumoral penetration.
Toxicity with specific liposomal preparations has also been a concern. Cationic liposomes elicit dose-dependent toxicity and pulmonary inflammation by promoting release of reactive oxygen intermediates, and this effect is more pronounced with multivalent cationic liposomes than monovalent cationic liposomes, such as N
-trimethylammonium methyl sulfate (51
). Neutral and negative liposomes do not seem to exhibit lung toxicity (52
). Cationic liposomes, while efficiently taking up nucleic acids, have had limited success for in vivo
gene down-regulation perhaps because of their stable intracellular nature and resultant failure to release siRNA contents. We selected DOPC because of its neutral properties and prior success in using this vehicle to deliver antisense oligonucleotides in vivo
In summary, we have shown that FAK siRNA using a neutral liposomal system is highly effective for down-regulating FAK expression in vivo. Furthermore, treatment with FAK siRNA-DOPC plus chemotherapy was highly effective in inhibiting ovarian cancer growth by both direct and indirect antivascular mechanisms. These findings suggest that FAK siRNA in combination with either docetaxel or cisplatin chemotherapy could be a potent therapeutic combination against ovarian cancer even in patients with chemotherapy-resistant tumors.