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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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PF271 enhanced the lethality of ABT-737.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).
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pone-0088587-g003: PF271 enhanced the lethality of ABT-737.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).

Mentions: To identify molecular targeted agents that exhibit a synergistic effect when co-administered with PF271, we treated cells with PF271 in combination with other inhibitors that target genes, particularly involved in the cell survival and apoptotic pathways such as the allosteric AKT inhibitor MK-2206, the PI3K/mTOR inhibitor BEZ235, the multiple kinase inhibitor sorafenib, and the BCL-2/BCL-XL inhibitor ABT-737. Drug combinations of PF271/MK-2206 or PF271/BEZ235 exhibited minor effects (data not shown). However, the combination of PF271/BEZ235/ABT-737 profoundly reduced cell growth and cell viability compared to the ABT-737 alone treatment (Fig. 3A). Sorafenib is known to down-regulate MCL1 [29]. Co-treatment of PF271/BEZ235 with sorafenib exhibited no significant effect compared to sorafenib alone (Fig. 3A). Notably, the RMGI and OVISE cell lines were at least 2-fold more sensitive to ABT-737 than the OVMANA and SKOV3 cell lines (Fig. 3A).


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)

PF271 enhanced the lethality of ABT-737.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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088587-g003: PF271 enhanced the lethality of ABT-737.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).
Mentions: To identify molecular targeted agents that exhibit a synergistic effect when co-administered with PF271, we treated cells with PF271 in combination with other inhibitors that target genes, particularly involved in the cell survival and apoptotic pathways such as the allosteric AKT inhibitor MK-2206, the PI3K/mTOR inhibitor BEZ235, the multiple kinase inhibitor sorafenib, and the BCL-2/BCL-XL inhibitor ABT-737. Drug combinations of PF271/MK-2206 or PF271/BEZ235 exhibited minor effects (data not shown). However, the combination of PF271/BEZ235/ABT-737 profoundly reduced cell growth and cell viability compared to the ABT-737 alone treatment (Fig. 3A). Sorafenib is known to down-regulate MCL1 [29]. Co-treatment of PF271/BEZ235 with sorafenib exhibited no significant effect compared to sorafenib alone (Fig. 3A). Notably, the RMGI and OVISE cell lines were at least 2-fold more sensitive to ABT-737 than the OVMANA and SKOV3 cell lines (Fig. 3A).

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