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Vav2 pharmaco-mimetic mice reveal the therapeutic value and caveats of the catalytic inactivation of a Rho exchange factor

Abstract

The current paradigm holds that the inhibition of Rho guanosine nucleotide exchange factors (GEFs), the enzymes that stimulate Rho GTPases, can be a valuable therapeutic strategy to treat Rho-dependent tumors. However, formal validation of this idea using in vivo models is still missing. In this context, it is worth remembering that many Rho GEFs can mediate both catalysis-dependent and independent responses, thus raising the possibility that the inhibition of their catalytic activities might not be sufficient per se to block tumorigenic processes. On the other hand, the inhibition of these enzymes can trigger collateral side effects that could preclude the practical implementation of anti-GEF therapies. To address those issues, we have generated mouse models to mimic the effect of the systemic application of an inhibitor for the catalytic activity of the Rho GEF Vav2 at the organismal level. Our results indicate that lowering the catalytic activity of Vav2 below specific thresholds is sufficient to block skin tumor initiation, promotion, and progression. They also reveal that the negative side effects typically induced by the loss of Vav2 can be bypassed depending on the overall level of Vav2 inhibition achieved in vivo. These data underscore the pros and cons of anti-Rho GEF therapies for cancer treatment. They also support the idea that Vav2 could represent a viable drug target.

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Fig. 1: Characterization of the L332A mutation of the Vav2 DH domain.
Fig. 2: Description and basic characterization of the Vav2L332A knock-in strain and other mouse models used in this study.
Fig. 3: Partial impairment of Vav2 GEF activity blocks skin tumorigenesis.
Fig. 4: The level of Vav2 catalytic activity inhibition determines the generation of negative side effects in mice.

References

  1. 1.

    Bustelo XR. RHO GTPases in cancer: known facts, open questions, and therapeutic challenges. Biochem Soc Trans. 2018;46:741–60.

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Vigil D, Cherfils J, Rossman KL, Der CJ. Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer. 2010;10:842–57.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6:167–80.

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Cullis J, Meiri D, Sandi MJ, Radulovich N, Kent OA, Medrano M, et al. The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1. Cancer Cell. 2014;25:181–95.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Fine B, Hodakoski C, Koujak S, Su T, Saal LH, Maurer M, et al. Activation of the PI3K pathway in cancer through inhibition of PTEN by exchange factor P-REX2a. Science. 2009;325:1261–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Heidary Arash E, Song KM, Song S, Shiban A, Attisano L. Arhgef7 promotes activation of the Hippo pathway core kinase Lats. EMBO J. 2014;33:2997–3011.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  7. 7.

    Robles-Valero J, Lorenzo-Martin LF, Fernandez-Pisonero I, Bustelo XR. Rho guanosine nucleotide exchange factors are not such bad guys after all in cancer. Small GTPases. 2018:1–7.https://doi.org/10.1080/21541248.2018.1423851.

  8. 8.

    Lyons LS, Burnstein KL. Vav3, a Rho GTPase guanine nucleotide exchange factor, increases during progression to androgen independence in prostate cancer cells and potentiates androgen receptor transcriptional activity. Mol Endocrinol. 2006;20:1061–72.

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Khanna N, Fang Y, Yoon MS, Chen J. XPLN is an endogenous inhibitor of mTORC2. Proc Natl Acad Sci USA. 2013;110:15979–84.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Robles-Valero J, Lorenzo-Martin LF, Menacho-Marquez M, Fernandez-Pisonero I, Abad A, Camos M, et al. A paradoxical tumor-suppressor role for the Rac1 exchange factor Vav1 in T cell acute lymphoblastic leukemia. Cancer Cell. 2017;32:608–23. e609

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Zandvakili I, Lin Y, Morris JC, Zheng Y. Rho GTPases: anti- or pro-neoplastic targets? Oncogene. 2017;36:3213–22.

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Svensmark JH, Brakebusch C. Rho GTPases in cancer: friend or foe? Oncogene. 2019;38:7447–56.

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Rodriguez-Fdez S, Bustelo XR. The Vav GEF family: an evolutionary and functional perspective. Cells. 2019;8:465–86.

    CAS  PubMed Central  Article  Google Scholar 

  14. 14.

    Bustelo XR. Vav family exchange factors: an integrated regulatory and functional view. Small GTPases. 2014;5:9.

    PubMed  Article  Google Scholar 

  15. 15.

    Menacho-Marquez M, Garcia-Escudero R, Ojeda V, Abad A, Delgado P, Costa C, et al. The Rho exchange factors Vav2 and Vav3 favor skin tumor initiation and promotion by engaging extracellular signaling loops. PLoS Biol. 2013;11:e1001615.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Citterio C, Menacho-Marquez M, Garcia-Escudero R, Larive RM, Barreiro O, Sanchez-Madrid F, et al. The Rho exchange factors Vav2 and Vav3 control a lung metastasis-specific transcriptional program in breast cancer cells. Sci Signal. 2012;5:ra71.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Lorenzo-Martin LF, Citterio C, Menacho-Marquez M, Conde J, Larive RM, Rodriguez-Fdez S, et al. Vav proteins maintain epithelial traits in breast cancer cells using miR-200c-dependent and independent mechanisms. Oncogene. 2019;38:209–27.

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Ruggiero C, Doghman-Bouguerra M, Sbiera S, Sbiera I, Parsons M, Ragazzon B, et al. Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion. Sci Signal. 2017;10:eaal2464.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Sauzeau V, Jerkic M, Lopez-Novoa JM, Bustelo XR. Loss of Vav2 proto-oncogene causes tachycardia and cardiovascular disease in mice. Mol Biol Cell. 2007;18:943–52.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Sauzeau V, Sevilla MA, Montero MJ, Bustelo XR. The Rho/Rac exchange factor Vav2 controls nitric oxide-dependent responses in mouse vascular smooth muscle cells. J Clin Investig. 2010;120:315–30.

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Fujikawa K, Iwata T, Inoue K, Akahori M, Kadotani H, Fukaya M, et al. VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans. PLoS ONE. 2010;5:e9050.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Rapley J, Tybulewicz VL, Rittinger K. Crucial structural role for the PH and C1 domains of the Vav1 exchange factor. EMBO Rep. 2008;9:655–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Chrencik JE, Brooun A, Zhang H, Mathews II, Hura GL, Foster SA, et al. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1. J Mol Biol. 2008;380:828–43.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Worthylake DK, Rossman KL, Sondek J. Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature. 2000;408:682–8.

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Liu X, Wang H, Eberstadt M, Schnuchel A, Olejniczak ET, Meadows RP, et al. NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio. Cell. 1998;95:269–77.

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Bustelo XR, Sauzeau V, Berenjeno IM. GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays. 2007;29:356–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995;81:1137–46.

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Crespo P, Bustelo XR, Aaronson DS, Coso OA, Lopez-Barahona M, Barbacid M, et al. Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav. Oncogene. 1996;13:455–60.

    CAS  PubMed  Google Scholar 

  29. 29.

    Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell. 1995;81:1159–70.

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Schuebel KE, Movilla N, Rosa JL, Bustelo XR. Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2. EMBO J. 1998;17:6608–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Wang Z, Pedersen E, Basse A, Lefever T, Peyrollier K, Kapoor S, et al. Rac1 is crucial for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyperactivation and hyperproliferation in vivo. Oncogene. 2010;29:3362–73.

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Garcia-Mariscal A, Li H, Pedersen E, Peyrollier K, Ryan KM, Stanley A, et al. Loss of RhoA promotes skin tumor formation and invasion by upregulation of RhoB. Oncogene. 2018;37:847–60.

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Abel EL, Angel JM, Kiguchi K, DiGiovanni J. Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications. Nat Protoc. 2009;4:1350–62.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Luch A. Nature and nurture—lessons from chemical carcinogenesis. Nat Rev Cancer. 2005;5:113–25.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Fabbiano S, Menacho-Marquez M, Sevilla MA, Albarran-Juarez J, Zheng Y, Offermanns S, et al. Genetic dissection of the Vav2-Rac1 signaling axis in vascular smooth muscle cells. Mol Cell Biol. 2014;34:4404–19.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. 36.

    Amado-Azevedo J, Valent ET, Van Nieuw, Amerongen GP. Regulation of the endothelial barrier function: a filum granum of cellular forces, Rho-GTPase signaling and microenvironment. Cell Tissue Res. 2014;355:557–76.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Garrett TA, Van Buul JD, Burridge K. VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2. Exp Cell Res. 2007;313:3285–97.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol. 2006;8:1223–34.

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Hunter SG, Zhuang G, Brantley-Sieders D, Swat W, Cowan CW, Chen J. Essential role of Vav family guanine nucleotide exchange factors in EphA receptor-mediated angiogenesis. Mol Cell Biol. 2006;26:4830–42.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Thomas EK, Cancelas JA, Chae HD, Cox AD, Keller PJ, Perrotti D, et al. Rac guanosine triphosphatases represent integrating molecular therapeutic targets for BCR-ABL-induced myeloproliferative disease. Cancer Cell. 2007;12:467–78.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Dutting S, Heidenreich J, Cherpokova D, Amin E, Zhang SC, Ahmadian MR, et al. Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets. J Thromb Haemost. 2015;13:827–38.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Malliri A, van der Kammen RA, Clark K, van der Valk M, Michiels F, Collard JG. Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours. Nature. 2002;417:867–71.

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Malliri A, Rygiel TP, van der Kammen RA, Song JY, Engers R, Hurlstone AF, et al. The rac activator Tiam1 is a Wnt-responsive gene that modifies intestinal tumor development. J Biol Chem. 2006;281:543–8.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Kawasaki Y, Tsuji S, Muroya K, Furukawa S, Shibata Y, Okuno M, et al. The adenomatous polyposis coli-associated exchange factors Asef and Asef2 are required for adenoma formation in Apc(Min/+)mice. EMBO Rep. 2009;10:1355–62.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Zugaza JL, Lopez-Lago MA, Caloca MJ, Dosil M, Movilla N, Bustelo XR. Structural determinants for the biological activity of Vav proteins. J Biol Chem. 2002;277:45377–92.

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Movilla N, Bustelo XR. Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins. Mol Cell Biol. 1999;19:7870–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Bouquier N, Fromont S, Zeeh JC, Auziol C, Larrousse P, Robert B, et al. Aptamer-derived peptides as potent inhibitors of the oncogenic RhoGEF Tgat. Chem Biol. 2009;16:391–400.

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Niebel B, Wosnitza CI, Famulok M. RNA-aptamers that modulate the RhoGEF activity of Tiam1. Bioorg Med Chem. 2013;21:6239–46.

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Verma R, Mohl D, Deshaies RJ. Harnessing the power of proteolysis for targeted protein inactivation. Mol Cell. 2020;77:446–60.

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Dotto GP, Rustgi AK. Squamous cell cancers: a unified perspective on biology and genetics. Cancer Cell. 2016;29:622–37.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Que SKT, Zwald FO, Schmults CD. Cutaneous squamous cell carcinoma: Incidence, risk factors, diagnosis, and staging. J Am Acad Dermatol. 2018;78:237–47.

    PubMed  Article  Google Scholar 

  52. 52.

    Huang Y, Zhao J, Mao G, Lee GS, Zhang J, Bi L, et al. Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas. Cell Discov. 2019;5:57.

    PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Doody GM, Bell SE, Vigorito E, Clayton E, McAdam S, Tooze R, et al. Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation. Nat Immunol. 2001;2:542–7.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Sauzeau V, Sevilla MA, Rivas-Elena JV, de Alava E, Montero MJ, Lopez-Novoa JM, et al. Vav3 proto-oncogene deficiency leads to sympathetic hyperactivity and cardiovascular dysfunction. Nat Med. 2006;12:841–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Barreira M, Fabbiano S, Couceiro JR, Torreira E, Martinez-Torrecuadrada JL, Montoya G, et al. The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteins. Sci Signal. 2014;7:ra35.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Blazquez for lab work and CIC facilities’ personnel for technical assistance. XRB is supported by grants from Worldwide Cancer Research (14-1248), the Castilla-León Government (CSI252P18, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (RTI2018-096481-B-I00), and the Spanish Association against Cancer (GC16173472GARC). XRB’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León autonomous government (CLC-2017-01). SF, SR-F, and LFL-M contracts have been supported by funding from the MSI (SF, BES-2010-031386; SR-F, BES-2013-063573), the Spanish Ministry of Universities (LFL-M, FPU13/02923), and the CLC-2017-01 grant (SR-F and LFL-M). JR-V has been supported by the CIBERONC and, currently, by the Spanish Association against Cancer. Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund.

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LFL-M participated in all the experiments shown in Figs. 3, 4b–e, in data analyses, and in artwork design. SF carried out the experiments shown in Figs. 1, 4a–e, analyzed the data, and contributed to artwork design. SR-F carried out the experiments shown in Figs. 2c–f, 4f, g, analyzed the data, and contributed to artwork design. AA helped in mouse work (crosses, genotyping, carcinogenesis experiments). MCG-M carried out the histopathological analyses of tumors. MD contributed to data analysis and paper writing. MC and JR-V contributed to the characterization of different phenotypic parameters of Vav2L332A mice. XRB conceived the work, analyzed data, wrote the paper, and performed the final editing of figures.

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Correspondence to Xosé R. Bustelo.

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Lorenzo-Martín, L.F., Rodríguez-Fdez, S., Fabbiano, S. et al. Vav2 pharmaco-mimetic mice reveal the therapeutic value and caveats of the catalytic inactivation of a Rho exchange factor. Oncogene 39, 5098–5111 (2020). https://doi.org/10.1038/s41388-020-1353-x

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