Targeted inhibition of FAK, PYK2 and BCL-XL synergistically enhances apoptosis in ovarian clear cell carcinoma cell lines.

Ovarian clear cell carcinoma (OCCC) displays a higher resistance to first line chemotherapy, requiring the development of new therapeutics. We previously identified a frequent chromosomal gain at 8q24 that harbors the focal-adhesion kinase (FAK) gene; the potential of this gene as a therapeutic target remains to be evaluated in OCCCs. We first examined the dependence of OCCCs on FAK and the PI3K/AKT signaling pathway. FAK was overexpressed in 20% of 67 OCCC samples, and this overexpression was correlated with its copy number gain. FAK copy number gains and mutations in PIK3CA accounted for about 40% of OCCC samples, suggesting that the FAK/PI3K/AKT axis is an attractive candidate for targeted therapeutics. We, therefore, treated ovarian cancer cell lines, including OCCC subtypes, with the FAK inhibitors PF-562,271 (PF271), and PF-573,228 (PF228). Ovarian cancer cells were more sensitive to PF271 than PF228. We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271. We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis. RMGI and OVISE cells were more sensitive to ABT-737 than OVMANA and SKOV3 cells, which have PIK3CA mutations. Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway. Therefore, PF271/ABT-737 treatment led to the inhibition of the anti-apoptotic proteins MCL1 and BCL-XL/BCL-2. We suggest that pharmacological inhibition of BCL-XL and FAK/PYK2 can be a potential therapeutic strategy for the treatment of OCCC

FAK and BCL2/BCL-XL inhibitors induced apoptosis.

<p>A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.</p

Drug responses of ovarian cancer cell lines to the FAK inhibitors.

<p>A, The chemical structure of the FAK inhibitors PF-573,228 (PF228) and PF-562,271 (PF271). B. OVISE cells were incubated for 24 hr at the indicated concentrations of the FAK inhibitors. Immunoblots were performed to assess inhibition of auto-phosphorylation by the FAK inhibitors. A vehicle control, containing dimethyl sulfoxide (DMSO), was performed. C, The viability of the ovarian cancer cells was determined after exposure to PF228 and PF271 for 72 hr. The results from only one experiment are shown; two additional studies also exhibited equivalent results.</p

FAK overexpression was associated with an increased copy number in OCCCs.

<p>A, Paraffin-embedded tumor sections were immunohistochemically stained using an anti-FAK antibody. The FAK staining results were scored into four categories based on the signal intensity: 0, no detection; 1, weak; 2, moderate; 3, strong. B, Variation in the FAK copy number was assessed with quantitative PCR. Copy number log ratios higher than 0.32 and 1.00 were considered evidence of copy number gain and gene amplification, respectively. The X-axis represents the samples. C, FAK overexpression (IHC score 3) correlated with the gains in copy number (copy number log ratio >0.32). Weak expressions of FAK (IHC score 1) resulted in normal or reduced FAK copy numbers (copy number log ratio <0.32). D, Samples with a mutation in <i>PIK3CA</i> (<i>PIK3CA</i>_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.</p

PF271 enhanced the lethality of ABT-737.

<p>A-upper panels, Ovarian cancer cell lines were exposed to various concentrations of ABT-737 (⋄: A), sorafenib (△: S), ABT-737 in combination with 1 µM BEZ235 and 5 µM PF271 (: ABP), or sorafenib in combination with 1 µM BEZ235 and 5 µM PF271 (▴: SBP) for 72 hr. Cell viability (%) was calculated and dose response curves were predicted with a three-parameter log-logistic function. A-lower panel, The EC₅₀ of the anti-apoptotic inhibitors alone or in combination. This represents the result of one experiment; two additional studies also exhibited equivalent results. B, The protein expression patterns of the anti-apoptotic proteins and the phosphorylation status of AKT in nine ovarian cancer cell lines. Immunoblots were done to assess the basal expression levels of BCL-2, BCL-XL, MCL1, and phosphorylated AKT. C, The standard mRNA expression levels of BCL-XL and BCL-2 in forty ovarian cancer cell lines (CCLE database).</p

FAK inhibitors down-regulate MCL1.

<p>A, MCL1 was transiently down-regulated by treatment with PF271 or PF228. RMGI and OVISE cells were incubated in the presence of PF271 or PF228 at 5 µM for RMGI and 10 µM for OVISE. The cells were harvested at the indicated time points and lysed. D indicates the dimethyl sulfoxide (DMSO) vehicle control. Immunoblots were performed for p-FAK, FAK, MCL1, AKT, and p-AKT. B, A proposed schematic model by how the combined pharmacological inhibition of FAK/PYK2 and BCL-XL/BCL-2 induces apoptosis.</p