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Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells


Mutations in RAS or BRAF are associated with poor prognosis and resistance to epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancer (CRC). Despite their common ability to activate downstream genes such as MEK and ERK, the therapeutic benefit of MEK inhibitors for patients with RAS/BRAF mutant CRC is limited, highlighting the need for biomarkers to predict the efficacy of MEK inhibition. Previously, we reported that a change in phosphorylation of ribosomal protein S6 (pS6) after MEK inhibition was significantly associated with sensitivity to MEK inhibition in gastric cancer cells. Here, we investigated the value of the response in pS6 for predicting the efficacy of trametinib, a MEK inhibitor, in patients with RAS/BRAF mutant CRC using patient-derived CRC organoids. We found that a subset of CRC cell lines and organoids were sensitive to trametinib. The change in phosphorylated ERK, a downstream molecule of the RAS/RAF/MEK pathway, was not significantly associated with trametinib sensitivity. On the other hand, only those with sensitivity showed a reduction of pS6 levels in response to trametinib. The change in pS6 after trametinib treatment was detectable by Western blotting, immunohistochemistry or immunocytochemistry. We also demonstrated an impact of MEK inhibition on pS6 in vivo using a xenograft model. Our data suggest that, in combination with patient-derived organoids, immunostaining-based detection of pS6 could be useful for prediction of trametinib sensitivity.

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Fig. 1: Suppression of pS6 after MEK inhibition is related to sensitivity to MEK inhibition in a subset of RAS/BRAF mutant CRC cell lines.
Fig. 2: Correlation between efficacy of MEK inhibition and pS6 response to treatment in CRC cells with acquired trametinib resistance.
Fig. 3: Establishment of patient-derived CRC organoids.
Fig. 4: Response of the pS6 level after MEK inhibition is also related to trametinib sensitivity in CRC organoids.

Data availability

The data that support the findings of this study are available from the corresponding authors, upon reasonable request.


  1. 1.

    Ciriello G, Miller ML, Aksoy BA, Senbabaoglu Y, Schultz N, Sander C. Emerging landscape of oncogenic signatures across human cancers. Nat Genet. 2013;45:1127–33.

    CAS  Article  Google Scholar 

  2. 2.

    Dancey JE, Bedard PL, Onetto N, Hudson TJ. The genetic basis for cancer treatment decisions. Cell. 2012;148:409–20.

    CAS  Article  Google Scholar 

  3. 3.

    Hyman DM, Taylor BS, Baselga J. Implementing Genome-Driven Oncology. Cell. 2017;168:584–99.

    CAS  Article  Google Scholar 

  4. 4.

    Hirashita Y, Tsukamoto Y, Yanagihara K, Fumoto S, Hijiya N, Nakada C, et al. Reduced phosphorylation of ribosomal protein S6 is associated with sensitivity to MEK inhibition in gastric cancer cells. Cancer Sci. 2016;107:1919–28.

    CAS  Article  Google Scholar 

  5. 5.

    Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011;141:1762–72.

    CAS  Article  Google Scholar 

  6. 6.

    Vlachogiannis G, Hedayat S, Vatsiou A, Jamin Y, Fernandez-Mateos J, Khan K, et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 2018;359:920–6.

    CAS  Article  Google Scholar 

  7. 7.

    Saito Y, Muramatsu T, Kanai Y, Ojima H, Sukeda A, Hiraoka N, et al. Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma. Cell Rep. 2019;27:1265–76.e1264.

    CAS  Article  Google Scholar 

  8. 8.

    Kondo, J & Inoue, M. Application of Cancer Organoid Model for Drug Screening and Personalized Therapy. Cells. 2019;8:470.

    CAS  Article  Google Scholar 

  9. 9.

    Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, et al. Cetuximab Monotherapy and Cetuximab plus Irinotecan in Irinotecan-Refractory Metastatic Colorectal Cancer. N Engl J Med. 2004;351:337–45.

    CAS  Article  Google Scholar 

  10. 10.

    Karapetis CS, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC, et al. K-ras Mutations and Benefit from Cetuximab in Advanced Colorectal Cancer. N Engl J Med. 2008;23:1757–65.

    Article  Google Scholar 

  11. 11.

    Sandhu J, Lavingia V, Fakih M. Systemic treatment for metastatic colorectal cancer in the era of precision medicine. J Surg Oncol. 2019;119:564–82.

    Article  Google Scholar 

  12. 12.

    Korphaisarn K, Kopetz S. BRAF-Directed Therapy in Metastatic Colorectal Cancer. Cancer J. 2016;22:175–8.

    CAS  Article  Google Scholar 

  13. 13.

    Wagner S, Vlachogiannis G, De Haven Brandon A, Valenti M, Box G, Jenkins L, et al. Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer. Oncogene. 2019;38:1717–33.

    CAS  Article  Google Scholar 

  14. 14.

    Cho M, Gong J, Frankel P, Synold TW, Lim D, Chung V, et al. A phase I clinical trial of binimetinib in combination with FOLFOX in patients with advanced metastatic colorectal cancer who failed prior standard therapy. Oncotarget. 2017;8:79750–60.

    Article  Google Scholar 

  15. 15.

    van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015;161:933–45.

    Article  Google Scholar 

  16. 16.

    Moriyama M, Tsukamoto Y, Fujiwara M, Kondo G, Nakada C, Baba T, et al. Identification of a novel human ankyrin-repeated protein homologous to CARP. Biochem Biophys Res Commun. 2001;285:715–23.

    CAS  Article  Google Scholar 

  17. 17.

    Jing J, Greshock J, Holbrook JD, Gilmartin A, Zhang X, McNeil E, et al. Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212. Mol Cancer Ther. 2012;11:720–9.

    CAS  Article  Google Scholar 

  18. 18.

    Yeh JJ, Routh ED, Rubinas T, Peacock J, Martin TD, Shen XJ, et al. KRAS/BRAF mutation status and ERK1/2 activation as biomarkers for MEK1/2 inhibitor therapy in colorectal cancer. Mol Cancer Ther. 2009;8:834–43.

    CAS  Article  Google Scholar 

  19. 19.

    Jacobi N, Seeboeck R, Hofmann E, Schweiger H, Smolinska V, Mohr T, et al. Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling. Oncotarget. 2017;8:107423–40.

    Article  Google Scholar 

  20. 20.

    Pickl M, Ries CH. Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene. 2009;28:461–468.

    CAS  Article  Google Scholar 

  21. 21.

    Fang JY, Richardson BC. The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 2005;6:322–7.

    CAS  Article  Google Scholar 

  22. 22.

    Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006;66:3992–5.

    CAS  Article  Google Scholar 

  23. 23.

    Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol. 2008;26:5705–12.

    Article  Google Scholar 

  24. 24.

    Corcoran RB, Andre T, Atreya CE, Schellens JHM, Yoshino T, Bendell JC, et al. Combined BRAF, EGFR, and MEK Inhibition in Patients with BRAF(V600E)-Mutant Colorectal Cancer. Cancer Discov. 2018;8:428–43.

    CAS  Article  Google Scholar 

  25. 25.

    Kopetz S, Grothey A, Tabernero J. Encorafenib, Binimetinib, and Cetuximab in BRAF V600E-Mutated Colorectal Cancer. Reply. N Engl J Med. 2020;382:877–8.

    PubMed  Google Scholar 

  26. 26.

    Corcoran RB, Atreya CE, Falchook GS, Kwak EL, Ryan DP, Bendell JC, et al. Combined BRAF and MEK Inhibition With Dabrafenib and Trametinib in BRAF V600-Mutant Colorectal Cancer. J Clin Oncol. 2015;33:4023–31.

    CAS  Article  Google Scholar 

  27. 27.

    Phan N, Hong JJ, Tofig B, Mapua M, Elashoff D, Moatamed NA, et al. A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids. Commun Biol. 2019;2:78.

    Article  Google Scholar 

  28. 28.

    Seidlitz T, Merker SR, Rothe A, Zakrzewski F, von Neubeck C, Grutzmann K, et al. Human gastric cancer modelling using organoids. Gut. 2019;68:207–17.

    CAS  Article  Google Scholar 

  29. 29.

    Zanoni M, Piccinini F, Arienti C, Zamagni A, Santi S, Polico R, et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep. 2016;6:19103.

    CAS  Article  Google Scholar 

  30. 30.

    Kim M, Mun H, Sung CO, Cho EJ, Jeon HJ, Chun SM, et al. Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nat Commun. 2019;10:3991.

    Article  Google Scholar 

  31. 31.

    Harris LA, Frick PL, Garbett SP, Hardeman KN, Paudel BB, Lopez CF, et al. An unbiased metric of antiproliferative drug effect in vitro. Nat Methods. 2016;13:497–500.

    CAS  Article  Google Scholar 

  32. 32.

    Hafner M, Niepel M, Chung M, Sorger PK. Growth rate inhibition metrics correct for confounders in measuring sensitivity to cancer drugs. Nat Methods. 2016;13:521–7.

    CAS  Article  Google Scholar 

  33. 33.

    Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007;12:9–22.

    CAS  Article  Google Scholar 

  34. 34.

    Dyachok J, Earnest S, Iturraran EN, Cobb MH, Ross EM. Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism. J Biol Chem. 2016;291:22414–26.

    CAS  Article  Google Scholar 

  35. 35.

    Munoz-Cordero MG, Lopez F, Garcia-Inclan C, Lopez-Hernandez A, Potes-Ares S, Fernandez-Vanes L, et al. Predictive value of EGFR-PI3K-pAKT-mTOR-pS6 pathway in sinonasal squamous cell carcinomas. Acta Otorrinolaringol Esp. 2019;70:16–24.

    Article  Google Scholar 

  36. 36.

    Chen CH, Hsia TC, Yeh MH, Chen TW, Chen YJ, Chen JT, et al. MEK inhibitors induce Akt activation and drug resistance by suppressing negative feedback ERK-mediated HER2 phosphorylation at Thr701. Mol Oncol. 2017;11:1273–87.

    CAS  Article  Google Scholar 

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We would like to thank Ms Mami Kimoto for their excellent assistance with experiments. We are also grateful to Hans Clevers, Tomohiro Mizutani, Else Driehuis and Stieneke van den Brink for critical advice on organoid experiments.


This work was supported at the Discretion of the President of Oita University (2019) and partly by JSPS KAKENHI Grant Number 18K15283.

Author information




Conceptualization: YH, YT; Formal analysis and investigation: YH, YK; Writing—original draft preparation: YH, YT; Writing—review and editing: DK, SK, NH, CN, TU, TH, KM, KH, TO, MK; Funding acquisition: YH; Resources: TH, TA, YU, HS, TE; Supervision: MI, MM, KM. All authors have approved the final version of the paper.

Corresponding authors

Correspondence to Yuka Hirashita or Yoshiyuki Tsukamoto.

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The authors declare no competing interests.

Ethical approval

This study was approved by the ethics committee of Oita University Faculty of Medicine (approval number: 1541).

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Hirashita, Y., Tsukamoto, Y., Kudo, Y. et al. Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells. Lab Invest (2021).

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