Cancer Chemotherapy and Pharmacology

, Volume 81, Issue 4, pp 745–754 | Cite as

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

  • Valentina Citi
  • Marzia Del Re
  • Alma Martelli
  • Vincenzo Calderone
  • Maria Cristina Breschi
  • Romano DanesiEmail author
Original Article



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.


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.


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.


mTOR/AKT/ERK pathway PTEN/PIK3CA mutations Everolimus Breast cancer Resistance 



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

Compliance with ethical standards

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.

Ethical approval

This work does not involve the use of human participants or animals.


  1. 1.
    Garcia Z, Kumar A, Marques M, Cortes I, Carrera AC (2006) Phosphoinositide 3-kinase controls early and late events in mammalian cell division. EMBO J 25(4):655–661. CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Zhang Y, Kwok-Shing Ng P, Kucherlapati M, Chen F, Liu Y, Tsang YH, de Velasco G, Jeong KJ, Akbani R, Hadjipanayis A, Pantazi A, Bristow CA, Lee E, Mahadeshwar HS, Tang J, Zhang J, Yang L, Seth S, Lee S, Ren X, Song X, Sun H, Seidman J, Luquette LJ, Xi R, Chin L, Protopopov A, Westbrook TF, Shelley CS, Choueiri TK, Ittmann M, Van Waes C, Weinstein JN, Liang H, Henske EP, Godwin AK, Park PJ, Kucherlapati R, Scott KL, Mills GB, Kwiatkowski DJ, Creighton CJ (2017) A pan-cancer proteogenomic atlas of PI3K/AKT/mTOR pathway alterations. Cancer Cell 31(6):820–832 e823. CrossRefPubMedGoogle Scholar
  3. 3.
    Atkins MB, Yasothan U, Kirkpatrick P (2009) Everolimus. Nat Rev Drug Discov 8(7):535–536. CrossRefPubMedGoogle Scholar
  4. 4.
    Shtivelband MI (2013) Everolimus in hormone receptor-positive advanced breast cancer: targeting receptor-based mechanisms of resistance. Breast 22(4):405–410. CrossRefPubMedGoogle Scholar
  5. 5.
    Hortobagyi GN, Chen D, Piccart M, Rugo HS, Burris HA 3rd, Pritchard KI, Campone M, Noguchi S, Perez AT, Deleu I, Shtivelband M, Masuda N, Dakhil S, Anderson I, Robinson DM, He W, Garg A, McDonald ER III, Bitter H, Huang A, Taran T, Bachelot T, Lebrun F, Lebwohl D, Baselga J (2015) Correlative analysis of genetic alterations and everolimus benefit in hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer: results From BOLERO-2. J Clin Oncol 34(5):419–426. CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Baselga J, Campone M, Piccart M, Burris HA 3rd, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI, Lebrun F, Beck JT, Ito Y, Yardley D, Deleu I, Perez A, Bachelot T, Vittori L, Xu Z, Mukhopadhyay P, Lebwohl D, Hortobagyi GN (2012) Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med 366(6):520–529. CrossRefPubMedGoogle Scholar
  7. 7.
    Karakas B, Bachman KE, Park BH (2006) Mutation of the PIK3CA oncogene in human cancers. Br J Cancer 94(4):455–459. CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Millis SZ, Ikeda S, Reddy S, Gatalica Z, Kurzrock R (2016) Landscape of phosphatidylinositol-3-kinase pathway alterations across 19784 diverse solid tumors. JAMA Oncol 2(12):1565–1573. CrossRefPubMedGoogle Scholar
  9. 9.
    Merenbakh-Lamin K, Ben-Baruch N, Yeheskel A, Dvir A, Soussan-Gutman L, Jeselsohn R, Yelensky R, Brown M, Miller VA, Sarid D, Rizel S, Klein B, Rubinek T, Wolf I (2013) D538G mutation in estrogen receptor-alpha: a novel mechanism for acquired endocrine resistance in breast cancer. Cancer Res 73(23):6856–6864. CrossRefPubMedGoogle Scholar
  10. 10.
    Fribbens C, O’Leary B, Kilburn L, Hrebien S, Garcia-Murillas I, Beaney M, Cristofanilli M, Andre F, Loi S, Loibl S, Jiang J, Bartlett CH, Koehler M, Dowsett M, Bliss JM, Johnston SR, Turner NC (2016) Plasma ESR1 mutations and the treatment of estrogen receptor-positive advanced breast cancer. J Clin Oncol 34(25):2961–2968. CrossRefPubMedGoogle Scholar
  11. 11.
    Jeselsohn R, Yelensky R, Buchwalter G, Frampton G, Meric-Bernstam F, Gonzalez-Angulo AM, Ferrer-Lozano J, Perez-Fidalgo JA, Cristofanilli M, Gomez H, Arteaga CL, Giltnane J, Balko JM, Cronin MT, Jarosz M, Sun J, Hawryluk M, Lipson D, Otto G, Ross JS, Dvir A, Soussan-Gutman L, Wolf I, Rubinek T, Gilmore L, Schnitt S, Come SE, Pusztai L, Stephens P, Brown M, Miller VA (2014) Emergence of constitutively active estrogen receptor-alpha mutations in pretreated advanced estrogen receptor-positive breast cancer. Clin Cancer Res 20(7):1757–1767. CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, Khuri FR (2005) Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 65(16):7052–7058. CrossRefPubMedGoogle Scholar
  13. 13.
    Brown RE, Buryanek J, Tammisetti VS, McGuire MF, Csencsits-Smith K (2016) Morphoproteomics and biomedical analytics confirm the mTORC2/Akt pathway as a resistance signature and activated ERK and STAT3 as concomitant prosurvival/antiapoptotic pathways in metastatic renal cell carcinoma (RCC) progressing on rapalogs: pathogenesis and therapeutic options. Oncotarget 7(27):41612–41621. CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Leung EY, Askarian-Amiri M, Finlay GJ, Rewcastle GW, Baguley BC (2015) Potentiation of growth inhibitory responses of the mTOR inhibitor everolimus by dual mTORC1/2 inhibitors in cultured breast cancer cell lines. PLoS One 10(7):e0131400. CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Mendoza MC, Er EE, Blenis J (2011) The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci 36(6):320–328. CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Gupta S, Ramjaun AR, Haiko P, Wang Y, Warne PH, Nicke B, Nye E, Stamp G, Alitalo K, Downward J (2007) Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 129(5):957–968. CrossRefPubMedGoogle Scholar
  17. 17.
    Ma L, Teruya-Feldstein J, Bonner P, Bernardi R, Franz DN, Witte D, Cordon-Cardo C, Pandolfi PP (2007) Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated mTOR activation in tuberous sclerosis and human cancer. Cancer Res 67(15):7106–7112. CrossRefPubMedGoogle Scholar
  18. 18.
    Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, Egia A, Sasaki AT, Thomas G, Kozma SC, Papa A, Nardella C, Cantley LC, Baselga J, Pandolfi PP (2008) Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest 118(9):3065–3074. PubMedCentralPubMedGoogle Scholar
  19. 19.
    Anavi-Goffer S, Baillie G, Irving AJ, Gertsch J, Greig IR, Pertwee RG, Ross RA (2012) Modulation of L-alpha-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids. J Biol Chem 287(1):91–104. CrossRefPubMedGoogle Scholar
  20. 20.
    Chan B, Cottrell JR, Li B, Larson KC, Ashford CJ, Levenson JM, Laeng P, Gerber DJ, Song J (2014) Development of a high-throughput AlphaScreen assay for modulators of synapsin I phosphorylation in primary neurons. J Biomol Screen 19(2):205–214. CrossRefPubMedGoogle Scholar
  21. 21.
    Garbison KE, Heinz BA, Lajiness ME, Weidner JR, Sittampalam GS (2004) Phospho-ERK Assays. In: Sittampalam GS, Coussens NP, Brimacombe K et al (eds) Assay Guidance Manual. Bethesda, RockvilleGoogle Scholar
  22. 22.
    Fallahi-Sichani M, Honarnejad S, Heiser LM, Gray JW, Sorger PK (2013) Metrics other than potency reveal systematic variation in responses to cancer drugs. Nat Chem Biol 9(11):708–714. CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Bader AG, Kang S, Zhao L, Vogt PK (2005) Oncogenic PI3K deregulates transcription and translation. Nat Rev Cancer 5(12):921–929. CrossRefPubMedGoogle Scholar
  24. 24.
    Oshiro C, Kagara N, Naoi Y, Shimoda M, Shimomura A, Maruyama N, Shimazu K, Kim SJ, Noguchi S (2015) PIK3CA mutations in serum DNA are predictive of recurrence in primary breast cancer patients. Breast Cancer Res Treat 150(2):299–307. CrossRefPubMedGoogle Scholar
  25. 25.
    Wikman H, Lamszus K, Detels N, Uslar L, Wrage M, Benner C, Hohensee I, Ylstra B, Eylmann K, Zapatka M, Sauter G, Kemming D, Glatzel M, Muller V, Westphal M, Pantel K (2012) Relevance of PTEN loss in brain metastasis formation in breast cancer patients. Breast Cancer Res 14(2):R49. CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Gonzalez-Angulo AM, Ferrer-Lozano J, Stemke-Hale K, Sahin A, Liu S, Barrera JA, Burgues O, Lluch AM, Chen H, Hortobagyi GN, Mills GB, Meric-Bernstam F (2011) PI3K pathway mutations and PTEN levels in primary and metastatic breast cancer. Mol Cancer Ther 10(6):1093–1101. CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Boscolo E, Coma S, Luks VL, Greene AK, Klagsbrun M, Warman ML, Bischoff J (2015) AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation. Angiogenesis 18(2):151–162. CrossRefPubMedGoogle Scholar
  28. 28.
    Hortobagyi GN, Chen D, Piccart M, Rugo HS, Burris HA III, Pritchard KI, Campone M, Noguchi S, Perez AT, Deleu I, Shtivelband M, Masuda N, Dakhil S, Anderson I, Robinson DM, He W, Garg A, McDonald ER III, Bitter H, Huang A, Taran T, Bachelot T, Lebrun F, Lebwohl D, Baselga J (2016) Correlative analysis of genetic alterations and everolimus benefit in hormone receptor-positive, Human epidermal growth factor receptor 2-negative advanced Breast cancer: results From BOLERO-2. J Clin Oncol 34(5):419–426. CrossRefPubMedGoogle Scholar
  29. 29.
    Moynahan ME, Chen D, He W, Sung P, Samoila A, You D, Bhatt T, Patel P, Ringeisen F, Hortobagyi GN, Baselga J, Chandarlapaty S (2017) Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR(+), HER2(-) advanced breast cancer: results from BOLERO-2. Br J Cancer 116(6):726–730. CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW, Schutte M (2007) Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines. Mol Cancer Res 5(2):195–201. CrossRefPubMedGoogle Scholar
  31. 31.
    O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N (2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66(3):1500–1508. CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Serra V, Scaltriti M, Prudkin L, Eichhorn PJ, Ibrahim YH, Chandarlapaty S, Markman B, Rodriguez O, Guzman M, Rodriguez S, Gili M, Russillo M, Parra JL, Singh S, Arribas J, Rosen N, Baselga J (2011) PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer. Oncogene 30(22):2547–2557. CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Soares HP, Ni Y, Kisfalvi K, Sinnett-Smith J, Rozengurt E (2013) Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells. PLoS One 8(2):e57289. CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Sinkala E, Sollier-Christen E, Renier C, Rosas-Canyelles E, Che J, Heirich K, Duncombe TA, Vlassakis J, Yamauchi KA, Huang H, Jeffrey SS, Herr AE (2017) Profiling protein expression in circulating tumour cells using microfluidic western blotting. Nat Commun 8:14622. CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Kallergi G, Agelaki S, Kalykaki A, Stournaras C, Mavroudis D, Georgoulias V (2008) Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients. Breast Cancer Res 10(5):R80. CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Bredemeier M, Kasimir-Bauer S, Kolberg HC, Herold T, Synoracki S, Hauch S, Edimiris P, Bankfalvi A, Tewes M, Kimmig R, Aktas B (2017) Comparison of the PI3KCA pathway in circulating tumor cells and corresponding tumor tissue of patients with metastatic breast cancer. Mol Med Rep 15(5):2957–2968. CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of PharmacyUniversity of PisaPisaItaly
  2. 2.Clinical Pharmacology and Pharmacogenetics Unit, Department of Clinical and Experimental MedicineUniversity of PisaPisaItaly

Personalised recommendations