Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy

Abstract

Purpose: Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC).

Experimental design: Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) were done using immunohistochemical analysis.

Results: A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 mug/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells.

Conclusions: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

Fig. 1

In vivo down-regulation of FAK by FAK siRNA. A, Western blot of lysates from tumor samples collected 0, 2, 4, and 6 days after a single administration of FAK siRNA or control siRNA incorporated in DOPC. Quantification of band intensity relative to β-actin. B, immunohistochemical staining for FAK expression (original magnification, ×200) after treatment with control siRNA-DOPC (a) or FAK siRNA-DOPC 2 (b), 4 (c), and 6 (d) days after a single dose.

Fig. 2

Therapeutic efficacy of FAK siRNA with docetaxel. Nude mice were injected i.p. with HeyA8 (A), SKOV3ip1 (B), or HeyA8MDR (C) cells and randomly allocated to one of the following groups, with therapy beginning 1 week after tumor cell injection: empty DOPC liposomes, control siRNA in DOPC, control siRNA in DOPC + docetaxel, FAK siRNA in DOPC, and FAK siRNA in DOPC + docetaxel. The animals were sacrificed when control mice became moribund (3–5 weeks after starting therapy) and necropsy was done. Left, columns, mean tumor weights; bars, SD. Right, individual weights.

Fig. 3

Therapeutic efficacy of FAK siRNA with cisplatin. Nude mice bearing A2780-CP20 tumors were randomly allocated to one of the following groups, with therapy beginning 1 week after tumor cell injection: empty liposomes, control siRNA in DOPC, control siRNA in DOPC + cisplatin, FAK siRNA in DOPC, and FAK siRNA in DOPC + cisplatin. The animals were sacrificed when control mice became moribund (4 weeks after tumor cell injection) and necropsy was done. Left, columns, mean tumor weights; bars, SD. Right, individual weights.

Fig. 4

A, immunohistochemistry (IHC) of FAK expression after long-term therapy in the HeyA8 orthotopic model. B, MVD was determined after immunohistochemical peroxidase staining for CD31. The number of vessels per ×100 field were counted as described in Materials and Methods. Representative slides from each group and average number of vessels per field. Five fields per slide and at least three slides per group were examined. VEGF (C) and matrix metalloproteinase-9 (MMP-9; D) immunohistochemical peroxidase staining was done on tumor sections obtained from each of the five therapy groups. E, representative images of immunofluorescence staining with CD31⁺ cells (red) and cells undergoing apoptosis (TUNEL stain; green). Yellow, endothelial cells (red) undergoing apoptosis (green). F, tumor sections from each group were stained for PCNA. The number of cancer cell nuclei that were strongly PCNA positive were counted and divided by the total number of cells. Representative sections from each group. Original magnification, ×100. Columns, mean percentage of PCNA-positive cells; bars, SD. Four fields per slide and at least three slides per group (all from different animals) were counted. G, immunofluorescence staining with TUNEL (green) for apoptosis and Hoechst (blue) for nuclei was done. Representative slides from each group. The number of apoptotic cells (green) was counted. Columns, mean number of TUNEL-positive cells; bars, SD. Four fields per slide and at least three slides per group (all from different animals) were counted. The columns in all graphs correspond to the labeled columns in the picture.