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

Yoon H, Choi YL, Song JY, Do I, Kang SY, Ko YH, Song S, Kim BG - PLoS ONE (2014)

Bottom Line: 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.Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Refractory Cancer Research, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
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.

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FAK overexpression was associated with an increased copy number in OCCCs.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 PIK3CA (PIK3CA_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.
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pone-0088587-g001: FAK overexpression was associated with an increased copy number in OCCCs.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 PIK3CA (PIK3CA_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.

Mentions: We first examined FAK protein expression levels in OCCC samples. More than 50% of the OCCC samples were positive for FAK expression, and about 21% of them showed strong FAK expression (Fig. 1A). We then assessed whether FAK overexpression was due to FAK copy number gain in OCCCs. FAK copy number gains and losses were estimated to have frequencies of 31% and 28% in the OCCC samples, respectively (Fig. 1B, Table S2 in File S1). In particular, 3 cases (4%) showed high level copy number gain, which were considered gene amplifications. The majority of the altered samples showed one copy gain or loss. FAK copy number gains significantly correlated with the strong protein expression (Fig. 1C, p-value = 0.0001). This suggests that FAK could be a selective target in a subset of OCCCs during tumorigenesis or for tumor progression. The fact that mutations in PIK3CA are common in OCCCs [2], [3] and that activated FAK sends signals via the PI3K/AKT signaling pathway suggests that PIK3CA mutations and FAK copy number gains might be mutually exclusive. Therefore, we screened the samples for PIK3CA mutations, which were found in 20% of the samples tested (Fig. 1D). PIK3CA mutations were found in some samples with a FAK copy number gain, suggesting that FAK copy number gains were not strictly mutually exclusive to PIK3CA mutations. Nonetheless, about 40% of the samples showed FAK copy number gain or PIK3CA mutation. This suggests that the FAK/PI3K/AKT axis might play an important role in OCCCs.


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

Yoon H, Choi YL, Song JY, Do I, Kang SY, Ko YH, Song S, Kim BG - PLoS ONE (2014)

FAK overexpression was associated with an increased copy number in OCCCs.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 PIK3CA (PIK3CA_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921183&req=5

pone-0088587-g001: FAK overexpression was associated with an increased copy number in OCCCs.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 PIK3CA (PIK3CA_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.
Mentions: We first examined FAK protein expression levels in OCCC samples. More than 50% of the OCCC samples were positive for FAK expression, and about 21% of them showed strong FAK expression (Fig. 1A). We then assessed whether FAK overexpression was due to FAK copy number gain in OCCCs. FAK copy number gains and losses were estimated to have frequencies of 31% and 28% in the OCCC samples, respectively (Fig. 1B, Table S2 in File S1). In particular, 3 cases (4%) showed high level copy number gain, which were considered gene amplifications. The majority of the altered samples showed one copy gain or loss. FAK copy number gains significantly correlated with the strong protein expression (Fig. 1C, p-value = 0.0001). This suggests that FAK could be a selective target in a subset of OCCCs during tumorigenesis or for tumor progression. The fact that mutations in PIK3CA are common in OCCCs [2], [3] and that activated FAK sends signals via the PI3K/AKT signaling pathway suggests that PIK3CA mutations and FAK copy number gains might be mutually exclusive. Therefore, we screened the samples for PIK3CA mutations, which were found in 20% of the samples tested (Fig. 1D). PIK3CA mutations were found in some samples with a FAK copy number gain, suggesting that FAK copy number gains were not strictly mutually exclusive to PIK3CA mutations. Nonetheless, about 40% of the samples showed FAK copy number gain or PIK3CA mutation. This suggests that the FAK/PI3K/AKT axis might play an important role in OCCCs.

Bottom Line: 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.Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Refractory Cancer Research, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus