Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Noncanonical hedgehog pathway activation through SRF–MKL1 promotes drug resistance in basal cell carcinomas

Abstract

Hedgehog pathway–dependent cancers can escape Smoothened (SMO) inhibition through mutations in genes encoding canonical hedgehog pathway components; however, around 50% of drug-resistant basal cell carcinomas (BCCs) lack additional variants of these genes. Here we use multidimensional genomics analysis of human and mouse drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factor serum response factor (SRF). Active SRF along with its coactivator megakaryoblastic leukemia 1 (MKL1) binds DNA near hedgehog target genes and forms a previously unknown protein complex with the hedgehog transcription factor glioma-associated oncogene family zinc finger-1 (GLI1), causing amplification of GLI1 transcriptional activity. We show that cytoskeletal activation through Rho and the formin family member Diaphanous (mDia) is required for SRF–MKL-driven GLI1 activation and for tumor cell viability. Remarkably, nuclear MKL1 staining served as a biomarker in tumors from mice and human subjects to predict tumor responsiveness to MKL inhibitors, highlighting the therapeutic potential of targeting this pathway. Thus, our study illuminates, for the first time, cytoskeletal-activation-driven transcription as a personalized therapeutic target for combatting drug-resistant malignancies.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The PTC53 BCC mouse model produces SMO-inhibitor-resistant tumors with human tumor characteristics.
Figure 2: Multicomponent genomic analyses identifies SRF as a previously unrecognized hedgehog cofactor with aberrant activation in rBCCs.
Figure 3: SRF and MKL1 are necessary for rBCC growth and potentiate hedgehog pathway activity.
Figure 4: MKL1 accumulates in the nucleus in mouse and human rBCCs.
Figure 5: Downstream hedgehog activation requires active Rho and mDia.
Figure 6: Pharmacological inhibition of MKL1 produces an in vivo therapeutic response in mouse and human BCCs.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Junttila, M.R. & de Sauvage, F.J. Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501, 346–354 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Ransohoff, K.J., Tang, J.Y. & Sarin, K.Y. Squamous change in basal-cell carcinoma with drug resistance. N. Engl. J. Med. 373, 1079–1082 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Atwood, S.X., Whitson, R.J. & Oro, A.E. Advanced treatment for basal cell carcinomas. Cold Spring Harb. Perspect. Med. 4, a013581 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Amaral, L. et al. Inhibitors of bacterial efflux pumps that also inhibit efflux pumps of cancer cells. Anticancer Res. 32, 2947–2957 (2012).

    CAS  PubMed  Google Scholar 

  5. Thomas-Schoemann, A. et al. Drug interactions with solid tumour–targeted therapies. Crit. Rev. Oncol. Hematol. 89, 179–196 (2014).

    Article  PubMed  Google Scholar 

  6. Nürnberg, A., Kitzing, T. & Grosse, R. Nucleating actin for invasion. Nat. Rev. Cancer 11, 177–187 (2011).

    Article  PubMed  CAS  Google Scholar 

  7. Barker, H.E., Paget, J.T., Khan, A.A. & Harrington, K.J. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat. Rev. Cancer 15, 409–425 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. McMillin, D.W., Negri, J.M. & Mitsiades, C.S. The role of tumour–stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discov. 12, 217–228 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Basset-Seguin, N., Sharpe, H.J. & de Sauvage, F.J. Efficacy of hedgehog pathway inhibitors in basal cell carcinoma. Mol. Cancer Ther. 14, 633–641 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Sekulic, A. et al. Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N. Engl. J. Med. 366, 2171–2179 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chang, A.L. & Oro, A.E. Initial assessment of tumor regrowth after vismodegib in advanced basal cell carcinoma. Arch. Dermatol. 148, 1324–1325 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Atwood, S.X. et al. Smoothened variants explain the majority of drug resistance in basal cell carcinoma. Cancer Cell 27, 342–353 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sharpe, H.J. et al. Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma. Cancer Cell 27, 327–341 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Atwood, S.X., Li, M., Lee, A., Tang, J.Y. & Oro, A.E. GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas. Nature 494, 484–488 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Atwood, S.X. et al. Rolling the genetic dice: neutral and deleterious smoothened mutations in drug-resistant basal cell carcinoma. J. Invest. Dermatol. 135, 2138–2141 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zaromytidou, A.I., Miralles, F. & Treisman, R. MAL and ternary complex factor use different mechanisms to contact a common surface on the serum response factor DNA-binding domain. Mol. Cell. Biol. 26, 4134–4148 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang, Z. et al. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature 428, 185–189 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Marais, R., Wynne, J. & Treisman, R. The SRF accessory protein Elk-1 contains a growth factor–regulated transcriptional activation domain. Cell 73, 381–393 (1993).

    Article  CAS  PubMed  Google Scholar 

  19. Janknecht, R. & Nordheim, A. Elk-1 protein domains required for direct and SRF-assisted DNA-binding. Nucleic Acids Res. 20, 3317–3324 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Miralles, F., Posern, G., Zaromytidou, A.I. & Treisman, R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113, 329–342 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Wang, G.Y. et al. Establishment of murine basal cell carcinoma allografts: a potential model for preclinical drug testing and for molecular analysis. J. Invest. Dermatol. 131, 2298–2305 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Webster, D.E. et al. Enhancer-targeted genome editing selectively blocks innate resistance to oncokinase inhibition. Genome Res. 24, 751–760 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bonilla, X. et al. Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma. Nat. Genet. 48, 398–406 (2016).

    Article  CAS  PubMed  Google Scholar 

  24. Olson, E.N. & Nordheim, A. Linking actin dynamics and gene transcription to drive cellular motile functions. Nat. Rev. Mol. Cell Biol. 11, 353–365 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Heidenreich, O. et al. MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo. J. Biol. Chem. 274, 14434–14443 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Zhang, H.M. et al. Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1. Nucleic Acids Res. 36, 2594–2607 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Blaker, A.L., Taylor, J.M. & Mack, C.P. PKA-dependent phosphorylation of serum response factor inhibits smooth muscle–specific gene expression. Arterioscler. Thromb. Vasc. Biol. 29, 2153–2160 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pawłowski, R., Rajakylä, E.K., Vartiainen, M.K. & Treisman, R. An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A. EMBO J. 29, 3448–3458 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Mokady, D. & Meiri, D. RhoGTPases—a novel link between cytoskeleton organization and cisplatin resistance. Drug Resist. Updat. 19, 22–32 (2015).

    Article  PubMed  Google Scholar 

  30. Guettler, S. et al. RPEL motifs link the serum response factor cofactor MAL but not myocardin to Rho signaling via actin binding. Mol. Cell. Biol. 28, 732–742 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Li, F. & Higgs, H.N. The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr. Biol. 13, 1335–1340 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Rath, N. & Olson, M.F. Rho-associated kinases in tumorigenesis: re-considering ROCK inhibition for cancer therapy. EMBO Rep. 13, 900–908 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Haak, A.J. et al. Targeting the myofibroblast genetic switch: inhibitors of myocardin-related transcription factor/serum response factor–regulated gene transcription prevent fibrosis in a murine model of skin injury. J. Pharmacol. Exp. Ther. 349, 480–486 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Sisson, T.H. et al. Inhibition of myocardin-related transcription factor/serum response factor signaling decreases lung fibrosis and promotes mesenchymal cell apoptosis. Am. J. Pathol. 185, 969–986 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ally, M.S. et al. Effects of combined treatment with arsenic trioxide and itraconazole in patients with refractory metastatic basal cell carcinoma. JAMA Dermatol. 152, 452–456 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Sekulic, A. et al. Pivotal ERIVANCE basal cell carcinoma (BCC) study: 12-month update of efficacy and safety of vismodegib in advanced BCC. J. Am. Acad. Dermatol. 72, 1021–1026. e8 (2015).

    Article  PubMed  Google Scholar 

  37. Vasudevan, H.N. & Soriano, P. SRF regulates craniofacial development through selective recruitment of MRTF cofactors by PDGF signaling. Dev. Cell 31, 332–344 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Esnault, C. et al. Rho–actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. Genes Dev. 28, 943–958 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schneider, P. et al. Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1. Nat. Commun. 6, 8023 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Horn, A. et al. Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis. Arthritis Rheum. 64, 2724–2733 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Parri, M. & Chiarugi, P. Rac and Rho GTPases in cancer cell motility control. Cell Commun. Signal 8, 23 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Jiang, P., Enomoto, A. & Takahashi, M. Cell biology of the movement of breast cancer cells: intracellular signalling and the actin cytoskeleton. Cancer Lett. 284, 122–130 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Morita, K., Lo Celso, C., Spencer-Dene, B., Zouboulis, C.C. & Watt, F.M. HAN11 binds mDia1 and controls GLI1 transcriptional activity. J. Dermatol. Sci. 44, 11–20 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Johnson, L.A. et al. Novel Rho/MRTF/SRF inhibitors block matrix-stiffness and TGF-β-induced fibrogenesis in human colonic myofibroblasts. Inflamm. Bowel Dis. 20, 154–165 (2014).

    Article  PubMed  Google Scholar 

  45. Wang, G.Y., Wang, J., Mancianti, M.L. & Epstein, E.H. Jr. Basal cell carcinomas arise from hair follicle stem cells in Ptch1+/− mice. Cancer Cell 19, 114–124 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kuleshov, M.V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44, W90–W97 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Peterson, K.A. et al. Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning. Genes Dev. 26, 2802–2816 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lee, E.Y. et al. Hedgehog pathway–regulated gene networks in cerebellum development and tumorigenesis. Proc. Natl. Acad. Sci. USA 107, 9736–9741 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Vokes, S.A., Ji, H., Wong, W.H. & McMahon, A.P. A genome-scale analysis of the cis-regulatory circuitry underlying sonic hedgehog–mediated patterning of the mammalian limb. Genes Dev. 22, 2651–2663 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sullivan, A.L. et al. Serum response factor utilizes distinct promoter- and enhancer-based mechanisms to regulate cytoskeletal gene expression in macrophages. Mol. Cell. Biol. 31, 861–875 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. McLean, C.Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Noubissi, F.K. et al. Role of CRD-BP in the growth of human basal cell carcinoma cells. J. Invest. Dermatol. 134, 1718–1724 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors wish to thank all members of the laboratory of A.E.O. and the Stanford Dermatology Department for suggestions and guidance. Specifically, the authors would like to thank S.P. Melo for guidance and assistance with ChIP–seq analyses. This work was funded by the V Foundation Translational Award, National Cancer Institute (R01CA157895), the National Institute of Arthritis and Musculoskeletal Disease (R01AR04786 and 5ARO54780), the Stanford Epithelial Biology Training Grant award to R.J.W. (T32-AR007422), National Institutes of Health (NIH) Pathway to Independence Award to S.X.A. (4R00CA17684703), and a Damon Runyon clinical investigatory award (J.Y.T.). The Stanford Cell Sciences Imaging Facility provided instrumentation and technical assistance for microscopy using a Leica SP8 confocal microscope funded by a National Center for Research Resources grant (1S10OD010580). The Stanford functional Genomics Facility provided sequencing services for ASZ RNA-seq and SRF ChIP–seq using the Illumina HiSeq 4000 platform purchased using an NIH S10 Shared Instrumentation Grant (S10OD018220).

Author information

Authors and Affiliations

Authors

Contributions

R.J.W. and A.E.O. designed the experiments and wrote the manuscript. R.J.W. performed the majority of experiments. A.L. performed all mouse tumor generation experiments except for rBCC drug treatment experiments with vismodegib and CCG-203971, which were administered by R.J.W. CCG-203971 in vivo drug treatment was repeated by M.A.F. The majority of cellular and molecular experiments were assisted and optimized by N.M.U. Exome- and RNA-seq analyses were carried out by J.R.L. and G.S. Co-IP experiments were carried out by A.M. C.Y.Y. assisted with SRF and MKL1 inhibition studies and knockdown studies as well as RHO and mDia experiments. S.X.A. assisted with RNA- and exome-seq library generation. S.Z.A., S.T.H., and K.Y.S. provided human tumor samples and annotation. E.H.E. and J.Y.T. designed the PTC53-BCC mouse resistance model. A.S.B., M.P.S., and E.Y.L. provided GLI ChIP data.

Corresponding author

Correspondence to Anthony E Oro.

Ethics declarations

Competing interests

A.E.O. is a clinical investigator funded by Novartis.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Whitson, R., Lee, A., Urman, N. et al. Noncanonical hedgehog pathway activation through SRF–MKL1 promotes drug resistance in basal cell carcinomas. Nat Med 24, 271–281 (2018). https://doi.org/10.1038/nm.4476

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4476

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer