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Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer

British Journal of Cancer volume 129, pages 1071–1082 (2023)Cite this article

Subjects

Abstract

Background

The identification of novel therapeutic strategies for metastatic colorectal cancer (mCRC) patients harbouring KRAS mutations represents an unmet clinical need. In this study, we aimed to clarify the role of p21-activated kinases (Paks) as therapeutic target for KRAS-mutated CRC.

Methods

Paks expression and activation levels were evaluated in a cohort of KRAS-WT or -mutated CRC patients by immunohistochemistry. The effects of Paks inhibition on tumour cell proliferation and signal transduction were assayed by RNAi and by the use of three pan-Paks inhibitors (PF-3758309, FRAX1036, GNE-2861), evaluating CRC cells, spheroids and tumour xenografts’ growth.

Results

Paks activation positively correlated with KRAS mutational status in both patients and cell lines. Moreover, genetic modulation or pharmacological inhibition of Paks led to a robust impairment of KRAS-mut CRC cell proliferation. However, Paks prolonged blockade induced a rapid tumour adaptation through the hyper-activation of the mTOR/p70S6K pathway. The addition of everolimus (mTOR inhibitor) prevented the growth of KRAS-mut CRC tumours in vitro and in vivo, reverting the adaptive tumour resistance to Paks targeting.

Conclusions

In conclusion, our results suggest the simultaneous blockade of mTOR and Pak pathways as a promising alternative therapeutic strategy for patients affected by KRAS-mut colorectal cancer.

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Fig. 1: Expression and activation of p21-activated kinases in CRC patients and cell lines.
Fig. 2: Effect of PAKs downregulation and pharmacological inhibition on cell proliferation and signalling activation in CRC cell lines.
Fig. 3: Prolonged exposure of HCT116 and SW480 CRC cells to Paks inhibitors led to a rapid tumor adaptation.
Fig. 4: Dissecting the effects of PAK1, 2 and 4 or KRAS knockdown on the mTOR pathway activation and cell proliferation.
Fig. 5: Overcoming CRC tumour adaptation to PF309 through mTOR inhibition.
Fig. 6: The combination of mTOR and Paks’ pathway blockade prevents HCT116 xenografts growth.

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Data availability

All relevant data are included in the manuscript and its Supplementary materials file or available from the corresponding author upon reasonable request.

References

  1. Biller LH, Schrag D. Diagnosis and treatment of metastatic colorectal cancer. JAMA]. 2021;325:669. https://jamanetwork.com/journals/jama/fullarticle/2776334.

    Article  CAS  PubMed  Google Scholar 

  2. Douillard J-Y, Oliner KS, Siena S, Tabernero J, Burkes R, Barugel M, et al. Panitumumab–FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013;369:1023–34. http://www.nejm.org/doi/10.1056/NEJMoa1305275.

    Article  CAS  PubMed  Google Scholar 

  3. Karthaus M, Hofheinz R-D, Mineur L, Letocha H, Greil R, Thaler J, et al. Impact of tumour RAS/BRAF status in a first-line study of panitumumab + FOLFIRI in patients with metastatic colorectal cancer. Br J Cancer. 2016;115:1215–22. http://www.nature.com/articles/bjc2016343.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Canon J, Rex K, Saiki AY, Mohr C, Cooke K, Bagal D, et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature. 2019;575:217–23. http://www.nature.com/articles/s41586-019-1694-1.

    Article  CAS  PubMed  Google Scholar 

  5. Skoulidis F, Li BT, Dy GK, Price TJ, Falchook GS, Wolf J, et al. Sotorasib for lung cancers with KRAS p.G12C mutation. N Engl J Med. 2021;384:2371–81. http://www.nejm.org/doi/10.1056/NEJMoa2103695.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rane CK, Minden A. P21 activated kinase signaling in cancer. Semin Cancer Biol. 2019;54:40–9.

    Article  CAS  PubMed  Google Scholar 

  7. Wang K, Baldwin GS, Nikfarjam M, He H. p21-activated kinase signalling in pancreatic cancer: new insights into tumour biology and immune modulation. World J Gastroenterol. 2018;24:3709–23. http://www.wjgnet.com/1007-9327/full/v24/i33/3709.htm.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shan L-H, Sun W-G, Han W, Qi L, Yang C, Chai C-C, et al. Roles of fibroblasts from the interface zone in invasion, migration, proliferation and apoptosis of gastric adenocarcinoma. J Clin Pathol. 2012;65:888–95.

    Article  CAS  PubMed  Google Scholar 

  9. Li Q, Zhang X, Wei N, Liu S, Ling Y, Wang H. p21-activated kinase 4 as a switch between caspase-8 apoptosis and NF-κB survival signals in response to TNF-α in hepatocarcinoma cells. Biochem Biophys Res Commun. 2018;503:3003–10. https://linkinghub.elsevier.com/retrieve/pii/S0006291X18317698.

    Article  CAS  PubMed  Google Scholar 

  10. Liu H, Liu K, Dong Z. The role of p21-activated kinases in cancer and beyond: where are we heading? Front Cell Dev Biol. 2021;9:641381. https://www.frontiersin.org/articles/10.3389/fcell.2021.641381/full.

  11. Wang Z, Fu M, Wang L, Liu J, Li Y, Brakebusch C, et al. P21-activated kinase 1 (PAK1) can promote ERK activation in a kinase-independent manner. J Biol Chem. 2013;288:20093–9.

  12. Won S-Y, Park J-J, Shin E-Y, Kim E-G. PAK4 signaling in health and disease: defining the PAK4–CREB axis. Exp Mol Med. 2019;51:1–9. https://www.nature.com/articles/s12276-018-0204-0.

    Article  CAS  PubMed  Google Scholar 

  13. Mauro CD, Pesapane A, Formisano L, Rosa R, D’Amato V, Ciciola P, et al. Urokinase-type plasminogen activator receptor (uPAR) expression enhances invasion and metastasis in RAS mutated tumors. Sci Rep. 2017;7:9388. http://www.nature.com/articles/s41598-017-10062-1.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Huang J, Dibble CC, Matsuzaki M, Manning BD. The TSC1-TSC2 complex is required for proper activation of mTOR complex 2. Mol Cell Biol. 2008;28:4104–15. https://journals.asm.org/doi/10.1128/MCB.00289-08.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 2006;441:424–30. http://www.nature.com/articles/nature04869.

    Article  CAS  PubMed  Google Scholar 

  16. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk. Cell. 2005;121:179–93. https://linkinghub.elsevier.com/retrieve/pii/S0092867405001984.

    Article  CAS  PubMed  Google Scholar 

  17. AlQurashi N, Gopalan V, Smith RA, Lam AKY. Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers. Hum Pathol. 2013;44:2089–96. https://linkinghub.elsevier.com/retrieve/pii/S0046817713001445.

    Article  CAS  PubMed  Google Scholar 

  18. Gulhati P, Bowen KA, Liu J, Stevens PD, Rychahou PG, Chen M, et al. mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res. 2011;71:3246–56. https://aacrjournals.org/cancerres/article/71/9/3246/575587/mTORC1-and-mTORC2-Regulate-EMT-Motility-and.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sudhan DR, Guerrero-Zotano A, Won H, González Ericsson P, Servetto A, Huerta-Rosario M, et al. Hyperactivation of TORC1 drives resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers. Cancer Cell. 2020;37:183–199.e5. https://linkinghub.elsevier.com/retrieve/pii/S1535610819305835.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li Y, Qiu X, Wang X, Liu H, Geck RC, Tewari AK, et al. FGFR-inhibitor-mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance. Nat Cell Biol. 2021;23:1187–98. https://www.nature.com/articles/s41556-021-00781-z.

    Article  CAS  PubMed  Google Scholar 

  21. Crawford JJ, Hoeflich KP, Rudolph J. p21-Activated kinase inhibitors: a patent review. Expert Opin Ther Pat. 2012;22:293–310. http://www.tandfonline.com/doi/full/10.1517/13543776.2012.668758.

    Article  CAS  PubMed  Google Scholar 

  22. Rudolph J, Murray LJ, Ndubaku CO, O’Brien T, Blackwood E, Wang W, et al. Chemically diverse group I p21-activated kinase (PAK) inhibitors impart acute cardiovascular toxicity with a narrow therapeutic window. J Med Chem. 2016;59:5520–41. https://pubs.acs.org/doi/10.1021/acs.jmedchem.6b00638.

    Article  CAS  PubMed  Google Scholar 

  23. Mpilla G, Aboukameel A, Muqbil I, Kim S, Beydoun R, Philip PA, et al. PAK4-NAMPT dual inhibition as a novel strategy for therapy resistant pancreatic neuroendocrine tumors. Cancers (Basel). 2019;11:1902. https://www.mdpi.com/2072-6694/11/12/1902.

    Article  CAS  PubMed  Google Scholar 

  24. Abu Aboud O, Chen C-H, Senapedis W, Baloglu E, Argueta C, Weiss RH. Dual and specific inhibition of NAMPT and PAK4 By KPT-9274 decreases kidney cancer growth. Mol Cancer Ther. 2016;15:2119–29. https://aacrjournals.org/mct/article/15/9/2119/92039/Dual-and-Specific-Inhibition-of-NAMPT-and-PAK4-By.

    Article  CAS  PubMed  Google Scholar 

  25. Khan HY, Uddin MH, Balasubramanian SK, Sulaiman N, Iqbal M, Chaker M, et al. PAK4 and NAMPT as novel therapeutic targets in diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma. Cancers (Basel). 2021;14:160. https://www.mdpi.com/2072-6694/14/1/160.

    Article  PubMed  Google Scholar 

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Funding

This work was supported by AIRC IG 21339 grant (RB); MFAG 21505 – 2018 grant (LF) and Lilly (LF); MFAG 27826 – 2022 grant (AS). DE was supported by AIRC fellowship for Italy – 26795.

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Author notes

  1. These authors contributed equally: Stefania Belli, Ada Pesapane.

Authors and Affiliations

  1. Department of Clinical Medicine and Surgery, University of Naples “Federico II”, 80131, Naples, Italy

    Stefania Belli, Ada Pesapane, Alberto Servetto, Daniela Esposito, Fabiana Napolitano, Claudia Maria Ascione, Alessandra Allotta, Luigi Formisano & Roberto Bianco

  2. CEINGE Biotecnologie Avanzate S.C.aR.L., Via G. Salvatore 486, 80145, Naples, Italy

    Nicola Zambrano

  3. Pathology Unit, University of Campania “L. Vanvitelli”, Naples, Italy

    Federica Zito Marino & Renato Franco

  4. Department of Precision Medicine, University of Campania “L. Vanvitelli”, Naples, Italy

    Teresa Troiani

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Contributions

Conceptualisation: SB, AP, LF and RB. Methodology: SB, AP, LF and RB. Writing—original draft preparation: SB, AP, AS LF. Investigation: SB, AP, DE, AA, FZM. Writing—review and editing: SB, AP, AS, DE, PC, CMA, AA, NZ, FZM, RF, TT, LF and RB. Supervision: LF and RB. Funding acquisition: AS, LF and RB. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Luigi Formisano or Roberto Bianco.

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Competing interests

AS reports honoraria from Eli Lilly, MSD, and Janssen and travel support from Bristol-Myers Squibb and AstraZeneca. LF declares the following competing interests: consultant and advisory board for Seagen, Amgen, BMS, MSD, Jansen and Pierre Fabre Pharma. RB declares the following competing interests: consultant and advisory board for BMS, MSD, Pfizer, AstraZeneca, Lilly and Novartis. The remaining authors declare no competing interests.

Ethics approval and consent to participate

CRC samples were retrospectively analysed at the Pathology Unit, University of Campania “L. Vanvitelli”. All patients agreed to participate in the study based on informed consent. Research Ethics Committee of the University of Campania “L. Vanvitelli” – AORN “Ospedale dei Colli” approved the study (reference n.0019530/2016). The study was performed in accordance with the Declaration of Helsinki.

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Belli, S., Pesapane, A., Servetto, A. et al. Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer. Br J Cancer 129, 1071–1082 (2023). https://doi.org/10.1038/s41416-023-02390-z

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