Advertisement

Investigational New Drugs

, Volume 32, Issue 4, pp 626–635 | Cite as

The combination of RAF265, SB590885, ZSTK474 on thyroid cancer cell lines deeply impact on proliferation and MAPK and PI3K/Akt signaling pathways

  • Susi Barollo
  • Loris Bertazza
  • Enke Baldini
  • Salvatore Ulisse
  • Elisabetta Cavedon
  • Marco Boscaro
  • Raffaele PezzaniEmail author
  • Caterina Mian
PRECLINICAL STUDIES

Summary

Papillary thyroid cancer (PTC) is the most frequent thyroid cancer entity, accounting for 88 % of cases. It may metastasize and loose iodine uptake capability, preventing any radioiodine or surgical treatment. The main gene altered in PTC is BRAF, which is found altered in over 50 % of cases. Moreover MAPK and PI3K/Akt pathways are greatly implicated in PTC development. Many target therapies for PTC are currently under investigation, unfortunately without the expected results. Aim of this study was to characterized the preclinical effectiveness of novel promising drugs, RAF265, SB590885 and ZSTK474 in 3 thyroid cancer cell lines (BCPAP, K1, 8505C). RAF265 and SB590885 target differentially BRAF, while ZSTK474 acts on PI3K. IC50 demonstrated high drug activities ranging from 0.1 to 6.2 μM, depending on drugs and cell type, while combination index revealed an interesting synergistic effect of combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474) in almost all cell lines. Moreover this synergistic effect was particularly evident by Western blot, whereas dual MAPK and PI3K/Akt inhibition was detected. In addition, treating cells with SB590885 induced marked morphological changes, leading to massive vacuolization. This suggests an activation of apoptotic process, as underlined by Annexin V flow cytometry analysis. Also cell cycle was altered in treated cells, without evidence of a common pattern, but rather with a more specific effect relying on single drug or combination regimen used. Since beneficial effects of in vitro combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474), it is recommended additional investigation. These data suggest the potential use of combination regimen in in vivo experiment or afterwards in human PTC.

Keywords

RAF265 SB590885 ZSTK474 BRAF Thyroid cancer 

Notes

Acknowledgments

This work was supported by Associazione Italiana per la Ricerca Oncologica di Base (AIROB, Padova, Italy). The authors thank Novartis for gift of RAF265.

Declaration of interests

The authors declare that they have no conflict of interest.

Supplementary material

10637_2014_108_MOESM1_ESM.pdf (2.8 mb)
Figure S1 (PDF 2822 kb)
10637_2014_108_MOESM2_ESM.pdf (4.4 mb)
Figure S2 (PDF 4466 kb)
10637_2014_108_MOESM3_ESM.pdf (3.4 mb)
Figure S3 (PDF 3450 kb)
10637_2014_108_MOESM4_ESM.pdf (3.6 mb)
Figure S4 (PDF 3715 kb)

References

  1. 1.
    Hundahl SA, Fleming ID, Fremgen AM, Menck HR (1998) A national cancer data base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985–1995 [see comments]. Cancer 83(12):2638–2648. doi: 10.1002/(SICI) CrossRefPubMedGoogle Scholar
  2. 2.
    Davies L, Welch HG (2006) Increasing incidence of thyroid cancer in the United States, 1973–2002. JAMA 295(18):2164–2167. doi: 10.1001/jama CrossRefPubMedGoogle Scholar
  3. 3.
    Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E, Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK (2008) Annual report to the nation on the status of cancer, 1975–2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 100(23):1672–1694. doi: 10.1093/jnci/djn389 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Tuttle RM, Lukes Y, Onstad L, Lushnikov E, Abrosimov A, Troshin V, Tsyb A, Davis S, Kopecky KJ, Francis G (2008) ret/PTC activation is not associated with individual radiation dose estimates in a pilot study of neoplastic thyroid nodules arising in Russian children and adults exposed to Chernobyl fallout. Thyroid 18(8):839–846. doi: 10.1089/thy.2008.0072 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Kondo T, Ezzat S, Asa SL (2006) Patho genetic mechanisms in thyroid follicular-cell neoplasia. Nat Rev Cancer 6(4):292–306. doi: 10.1038/nrc1836 CrossRefPubMedGoogle Scholar
  6. 6.
    Xing M (2008) Recent advances in molecular biology of thyroid cancer and their clinical implications. Otolaryngol Clin N Am 41(6):1135–1146. doi: 10.1016/j.otc.2008.07.001 CrossRefGoogle Scholar
  7. 7.
    Ciampi R, Nikiforov YE (2005) Alterations of the BRAF gene in thyroid tumors. Endocr Pathol 16(3):163–172CrossRefPubMedGoogle Scholar
  8. 8.
    Barollo S, Pezzani R, Cristiani A, Redaelli M, Zambonin L, Rubin B, Bertazza L, Zane M, Mucignat-Caretta C, Bulfone A, Pennelli G, Casal Ide E, Pelizzo MR, Mantero F, Moro S, Mian C (2014) Prevalence, tumorigenic role, and biochemical implications of rare BRAF alterations. Thyroid. doi: 10.1089/thy.2013.0403 PubMedGoogle Scholar
  9. 9.
    Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle P, Elder DE, Herlyn M (2003) Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res 63(4):756–759PubMedGoogle Scholar
  10. 10.
    Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R (2004) Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116(6):855–867CrossRefPubMedGoogle Scholar
  11. 11.
    Poulikakos PI, Solit DB (2011) Resistance to MEK inhibitors: should we co-target upstream? Sci Signal 4(166):16. doi: 10.1126/scisignal.2001948 CrossRefGoogle Scholar
  12. 12.
    King AJ, Patrick DR, Batorsky RS, Ho ML, Do HT, Zhang SY, Kumar R, Rusnak DW, Takle AK, Wilson DM, Hugger E, Wang L, Karreth F, Lougheed JC, Lee J, Chau D, Stout TJ, May EW, Rominger CM, Schaber MD, Luo L, Lakdawala AS, Adams JL, Contractor RG, Smalley KS, Herlyn M, Morrissey MM, Tuveson DA, Huang PS (2006) Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res 66(23):11100–11105. doi: 10.1158/0008-5472 CrossRefPubMedGoogle Scholar
  13. 13.
    Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, Hirono S, Yamazaki K, Yamori T (2006) Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J Natl Cancer Inst 98(8):545–556. doi: 10.1093/jnci/djj133 CrossRefPubMedGoogle Scholar
  14. 14.
    Pezzani R, Rubin B, Redaelli M, Radu C, Barollo S, Cicala MV, Salva M, Mian C, Mucignat-Caretta C, Simioni P, Iacobone M, Mantero F (2014) The antiproliferative effects of ouabain and everolimus on adrenocortical tumor cells. Endocr J 61(1):41–53CrossRefPubMedGoogle Scholar
  15. 15.
    Mariniello B, Rosato A, Zuccolotto G, Rubin B, Cicala MV, Finco I, Iacobone M, Frigo AC, Fassina A, Pezzani R, Mantero F (2012) Combination of sorafenib and everolimus impacts therapeutically on adrenocortical tumor models. Endocr Relat Cancer 19(4):527–539. doi: 10.1530/ERC-11-0337 CrossRefPubMedGoogle Scholar
  16. 16.
    Pilli T, Prasad KV, Jayarama S, Pacini F, Prabhakar BS (2009) Potential utility and limitations of thyroid cancer cell lines as models for studying thyroid cancer. Thyroid 19(12):1333–1342. doi: 10.1089/thy.2009.0195 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Aksamitiene E, Kiyatkin A, Kholodenko BN (2012) Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem Soc Trans 40(1):139–146. doi: 10.1042/BST20110609 CrossRefPubMedGoogle Scholar
  18. 18.
    Gonzalez-Polo RA, Boya P, Pauleau AL, Jalil A, Larochette N, Souquere S, Eskelinen EL, Pierron G, Saftig P, Kroemer G (2005) The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. J Cell Sci 118(14):3091–3102. doi: 10.1242/jcs.02447 CrossRefPubMedGoogle Scholar
  19. 19.
    Su Y, Vilgelm AE, Kelley MC, Hawkins OE, Liu Y, Boyd KL, Kantrow S, Splittgerber RC, Short SP, Sobolik T, Zaja-Milatovic S, Dahlman KB, Amiri KI, Jiang A, Lu P, Shyr Y, Stuart DD, Levy S, Sosman JA, Richmond A (2012) RAF265 inhibits the growth of advanced human melanoma tumors. Clin Cancer Res 18(8):2184–2198. doi: 10.1158/1078-0432 PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Saiselet M, Floor S, Tarabichi M, Dom G, Hebrant A, van Staveren WC, Maenhaut C (2012) Thyroid cancer cell lines: an overview. Front Endocrinol 3:133. doi: 10.3389/fendo.2012.00133 CrossRefGoogle Scholar
  21. 21.
    Vianello F, Mazzarotto R, Mian C, Lora O, Saladini G, Servodio O, Basso M, Pennelli G, Pelizzo MR, Sotti G (2012) Clinical outcome of low-risk differentiated thyroid cancer patients after radioiodine remnant ablation and recombinant human thyroid-stimulating hormone preparation. Clin Oncol 24(3):162–168. doi: 10.1016/j.clon.2011.02.011 CrossRefGoogle Scholar
  22. 22.
    Dan S, Okamura M, Mukai Y, Yoshimi H, Inoue Y, Hanyu A, Sakaue-Sawano A, Imamura T, Miyawaki A, Yamori T (2012) ZSTK474, a specific phosphatidylinositol 3-kinase inhibitor, induces G1 arrest of the cell cycle in vivo. Eur J Cancer 48(6):936–943. doi: 10.1016/j.ejca.2011.10.006 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Susi Barollo
    • 1
  • Loris Bertazza
    • 1
  • Enke Baldini
    • 2
  • Salvatore Ulisse
    • 2
  • Elisabetta Cavedon
    • 1
  • Marco Boscaro
    • 1
  • Raffaele Pezzani
    • 1
    Email author
  • Caterina Mian
    • 1
  1. 1.Endocrinology Unit, Department of MedicineUniversity of PaduaPaduaItaly
  2. 2.Department of Experimental Medicine“Sapienza” University of RomeRomeItaly

Personalised recommendations