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. 2013 Jun;30(5):579-94.
doi: 10.1007/s10585-012-9562-5. Epub 2012 Dec 30.

Inhibition of Focal Adhesion Kinase (FAK) Activity Prevents Anchorage-Independent Ovarian Carcinoma Cell Growth and Tumor Progression

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Free PMC article

Inhibition of Focal Adhesion Kinase (FAK) Activity Prevents Anchorage-Independent Ovarian Carcinoma Cell Growth and Tumor Progression

Kristy K Ward et al. Clin Exp Metastasis. .
Free PMC article

Abstract

Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the peri-ovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer.

Figures

Figure 1
Figure 1
Elevated FAK gene (Ptk2) amplification in ovarian cancer and Kaplan-Meier analyses of FAK mRNA levels with overall patient survival. (A) The cBio Cancer Genomics Portal (http://www.cbioportal.org/public-portal/) was queried for percentage of Ptk2 gene amplification in The Cancer Genome Atlas (TCGA) with different cancers and significance determined by the Chi-squared test (*** p<0.001). (B) The Kaplan-Meier Plotter (http://www.kmplot.com/ovar) was queried to evaluate Affymetrix microarray expression of FAK mRNA levels in 961 annotated serous ovarian cancer patient tumor samples. Selections were: overall survival (follow up threshold of 10 years), split patients by median, stage (all), histology (serous), grade (all), debulk (all), and chemotherapy treatments (all). High levels of FAK expression (red) are associated with decreased patient survival (logrank P = 0.0007) and the Hazard ratio (with 95% confidence intervals) is shown.
Figure 2
Figure 2
In vivo passage of ID8 murine ovarian carcinoma cells in C57Bl6 mice result in the spontaneous generation of aggressive cells. (A) Schematic summarizing the intraperitoneal (IP) injection of ID8 cells, re-isolation from ascites after 43 days, growth and expansion in anchorage-independent (poly-HEMA-coated plates) for 4 weeks with the resulting pooled population of cells termed ID8-IP. (B) Adherent proliferation of 50,000 ID8 or ID8-IP cells over 6 days. (C) Anchorage-independent growth of 250,000 ID8 or ID8-IP cells on poly-HEMA coated plates over 6 days. (B and C) Values are means (+/- SD) of triplicate points, (** p<0.01, *** p<0.001). (D) Phase contrast images of ID8 or ID8-IP cells under adherent and confluent (Day 5) or suspended conditions (Day 6). Scale is 70 μM. (E) Soft agar colony number of ID8 or ID8-IP cells after 7 days. Values are means (+/- SD) of triplicate points, (** p<0.01). Scale is 3 mm. (F) Cell lysates were prepared from ID8 and ID8-IP cells growing under adherent or suspended conditions after 3 days. Total protein was normalized for actin and E-cadherin blotting was performed on the same membrane. Phospho-specific immunoblotting for FAK Y397 (pY397) or c-Src Y416 (pY416) phosphorylation was performed and the membranes were re-probed for total FAK or c-Src, respectively.
Figure 3
Figure 3
ID8-IP ovarian carcinoma cells exhibit enhanced orthotopic tumor growth and metastasis. mCherry-expressing ID8 and ID8-IP cells (500,000 cells in 7 μL of Matrigel) were injected into the ovarian bursa of 8-10 week old C57Bl6 mice and evaluated after 28 days. (A) Representative images of combined mCherry-fluorescence with brightfield images of opened peritoneal cavity. Arrows (yellow) indicate positions of ovaries injected with ID8 or ID8-IP tumor cells. Scale is 0.5 cm. (B) Representative images of surgically extracted ID8 or ID8-IP ovarian tumors with uterine horns. Scale is 1 mm divisions. (C) Primary tumor weights of ID8 and ID8-IP injected mice. (D) Ascites was collected, peritoneal cavity washed with saline, and total mCherry-positive cells enumerated by flow cytometry. (E) Peritoneal-associated metastatic tumor sites were quantified by counting mCherry-positive nodules visualized by OV100 imaging. (C-E) Values are means (+/- SD) from ID8 (n=9) or ID8-IP (n=8) and represent one of two independent experiments (* p<0.05, *** p<0.001).
Figure 4
Figure 4
Ovary-associated ID8 and ID8-IP tumors. (A) Representative x40 magnification of paraffin-embedded H&E-stained ID8 or (B) ID8-IP tumors 28 days after orthotopic injection. Right, is H&E x200 magnification (boxed region of x40 image) and a corresponding serial obtained tumor section stained with antibodies to dsRed (diaminobenzidine, DAB) and haematoxylin. The area of ID8-IP infiltration into the ovary is circled (green). The T, tumor; F, follicle, and CL, corpus luteum. Scale is 100 μm.
Figure 5
Figure 5
PF-271 FAK inhibition prevents anchorage-independent ID8-IP growth without effects on c-Src Y416 phosphorylation. (A) Adherent proliferation of ID8 or ID8-IP cells over 6 days in the presence of vehicle (dimethylsulfoxide, DMSO) or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are average fold-change above 50,000 starting cells from triplicate points (+/- SD), (***p<0.001). (B) Anchorage-independent growth of 250,000 ID8-IP cells on poly-HEMA coated plates over 5 days in the presence of DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are means (+/- SD) of triplicate points, (*p<0.05, *** p<0.001). (C) ID8-IP soft agar colony number after 7 days in the presence of DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM). Values are means (+/- SD) of triplicate points, (*p<0.05, *** p<0.001). (D) Protein lysates of anchorage-independent ID8-IP cells on poly-HEMA plates treated with DMSO or increasing concentrations of PF-271 (0.1 to 1.0 μM) were evaluated by immunoblotting for FAK Y397 phosphorylation (pY397), total FAK, c-Src Y416 phosphorylation (pY416 Src), total c-Src, and total actin levels.
Figure 6
Figure 6
Oral administration of PF-271 FAK inhibitor prevents ID8-IP tumor growth and metastasis independent of effects on c-Src Y416 phosphorylation. (A) mCherry-labeled ID8-IP tumor cells were bursal-injected and after 7 days, vehicle or PF-271 (30 mg/kg) were administered by oral gavage twice-daily (BID). Mice were euthanized after 28 days and (A) primary tumor weight was determined for mice treated with vehicle (n=8) or PF-271 (n=10). (B) Peritoneal-associated metastatic tumor sites were quantified by counting mCherry-positive nodules visualized by OV100 imaging. (A and B) Values are means (+/- SD) (* p<0.05, ** p<0.01). (C) Evaluation of FAK Y397 phosphorylation (pY397), total FAK, c-Src Y416 phosphorylation (pY416 Src), total c-Src, and total actin levels by immunoblotting using normal ovary tissue or ID8-IP tumors from 5 independent vehicle- or PF-271-treated mice. (D) Ratio of pY397 phosphorylated FAK to total FAK levels normalized to normal ovary (set to 1) and determined by densitometry using Image J (n= 5 per group, *** p<0.001). (E) Ratio of pY416 phosphorylated c-Src to total c-Src levels normalized to normal ovary (set to 1) and determined by densitometry using Image J (n= 5 per group).
Figure 7
Figure 7
Alterations in ID8-IP tumor-associated endothelial cells and tumor apoptosis in PF-271-treated mice. (A) Representative x40 images of paraffin-embedded H&E stained tumors from vehicle- or PF-271-treated mice. Right, boxed region x40 image at x200 magnification. Scale is 100 μM. (B) Representative fluorescent microscopy images of anti-CD31 antibody (green) and cell nuclei (Hoechst, blue) staining within ID8-IP tumors from vehicle- or PF-271-treated mice. (C) Fluorescent images from five random fields (at 10X) from 3 different ID8-IP tumors from vehicle- or PF-271-treated mice were acquired and the number of CD31-positive cells per field were enumerated using Image J. Values are means (+/- SD) (*p<0.05). (D) Representative fluorescent microscopy images of TUNEL (green) and cell nuclei (Hoechst, blue) staining within ID8-IP tumors from vehicle- or PF-271-treated mice. Scale is 100 μM. (E) Images were acquired and enumerated as above. Values are means (+/- SD) (*p<0.05).
Figure 8
Figure 8
Genetic support for the importance of FAK expression and activity in ID8-IP anchorage-independent cell survival. (A) Stable ID8-IP FAK shRNA knockdown cells were transiently reconstituted with GFP-FAK WT or GFP-FAK kinase-dead (KD), sorted for GFP-expression, and pooled populations of cells expanded and then used in cell proliferation assays within 10-14 days. Flow cytometry shows GFP (open histogram) or background cell fluorescence (dark histogram) of GFP-FAK WT and GFP-FAK KD cells after expansion. (B) Immunoblotting of ID8-IP cells lysates from scrambled shRNA (Scr), FAK shRNA-expressing, and FAK shRNA-expressing reconstituted with GFPFAK WT or GFP-FAK KD using antibodies to FAK and actin as a loading control. GFPFAK (150 kDa) and endogenous FAK (115 kDa) are denoted. (C) Adherent cell viability and proliferation of 50,000 ID8-IP cells (Day 0) expressing Scr shRNA, FAK shRNA, and FAK shRNA cells reconstituted with GFP-FAK WT or GFP-FAK KD over 5 days as measured by ViCellXR enumeration. Values are means (+/- SD) of triplicate points (**p<0.01). (D) Suspended cell viability and survival of 250,000 ID8-IP cells (Day 0) expressing Scr shRNA, FAK shRNA, and FAK shRNA cells reconstituted with GFP-FAK WT or GFP-FAK KD over 5 days as measured by ViCellXR enumeration. Values are means (+/- SD) of triplicate points plotted as percent of initial cell number (**p<0.01, ***p<0.001).

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