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Phosphorylation of AKT and ERK1/2 and mutations of PIK3CA and PTEN are predictive of breast cancer cell sensitivity to everolimus in vitro

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Abstract

Background

Everolimus is the hydroxyethyl derivative of sirolimus and a strong inhibitor of mammalian target of rapamycin (mTOR). This drug has immunosuppressive and anticancer activities and the present in vitro study was aimed at identifying the cellular and molecular profiles of breast cancer cells predictive of sensitivity to everolimus.

Materials and methods

MCF-7, T-47D, ZR-75-1, CAMA-1, HCC-1500 and MCF-10A cells were used and viability was assessed using WST-1 dye. Sensitivity to everolimus was correlated with phosphorylation of AKT (Ser473/Thr308), mTOR (Ser2448), and ERK1/2 (Thr202/Tyr204) and mutational profile of KRAS, NRAS, BRAF, PIK3CA, PTEN, TSC1, TSC2 and FRAP genes. Protein phosphorylation was evaluated by AlphaScreen SureFire, while the mutational status was examined by digital droplet PCR and Sanger sequencing.

Results

Everolimus showed a transient growth inhibition in non-tumorigenic cells, while in tumorigenic lines the drug suppressed the proliferation in a concentration-dependent manner but with different potency (IC50) and efficacy (Emax), being ZR-75-1 the most sensitive and T47D the least sensitive. MCF-7, T47D and HCC1500 had activating mutations in PIK3CA gene, while loss-of-activity PTEN mutations were detected in sensitive cell lines, including ZR-75-1, which showed no changes or minimal increase in the amount of p-AKT(Ser473/Thr308) and p-ERK1/2(Thr202/Tyr204) induced by everolimus compared to the resistant cell line T47D in which phosphorylation of AKT and ERK was increased.

Conclusions

Cellular levels of p-AKT(Ser473/Thr308) and p-ERK1/2(Thr202/Tyr204), activating mutations of PIK3CA and inactivating mutations of PTEN may predict response to everolimus in breast cancer cells; these findings have potential applications for treatment personalization of everolimus in breast cancer patients.

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Funding

This article was funded by an unrestricted grant from Novartis Pharma (Italy) to R. Danesi.

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Authors and Affiliations

  1. Department of Pharmacy, University of Pisa, Pisa, Italy

    Valentina Citi, Alma Martelli, Vincenzo Calderone & Maria Cristina Breschi

  2. Clinical Pharmacology and Pharmacogenetics Unit, Department of Clinical and Experimental Medicine, University of Pisa, 55, Via Roma, 56126, Pisa, Italy

    Marzia Del Re & Romano Danesi

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  1. Valentina Citi
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  2. Marzia Del Re
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Correspondence to Romano Danesi.

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Conflict of interest

R. Danesi has received research grants from Novartis, Pfizer, AstraZeneca. R. Danesi has received a speaker honorarium from Celgene, Pfizer, BMS, MSD, Roche, Sanofi, Lilly, Janssen. All other Authors declare no conflict of interest.

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Citi, V., Del Re, M., Martelli, A. et al. Phosphorylation of AKT and ERK1/2 and mutations of PIK3CA and PTEN are predictive of breast cancer cell sensitivity to everolimus in vitro. Cancer Chemother Pharmacol 81, 745–754 (2018). https://doi.org/10.1007/s00280-018-3543-6

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