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

Nature Medicine volume 24, pages 271–281 (2018)Cite this article

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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.

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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.

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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).

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Authors and Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Ramon J Whitson, Amar Mirza, Catherine Y Yao, Alexander S Brown, Jiang R Li, Gautam Shankar, Scott X Atwood, Kavita Y Sarin, Jean Y Tang & Anthony E Oro

  2. Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

    Ramon J Whitson, Nicole M Urman, Amar Mirza, Catherine Y Yao, Alexander S Brown, Jiang R Li, Gautam Shankar, Scott X Atwood, Eunice Y Lee, S Tyler Hollmig, Sumaira Z Aasi, Kavita Y Sarin, Matthew P Scott, Jean Y Tang & Anthony E Oro

  3. Children's Hospital Oakland Research Institute, Oakland, California, USA

    Alex Lee, Micah A Fry & Ervin H Epstein Jr

  4. Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA

    Scott X Atwood

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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.

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Correspondence to Anthony E Oro.

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Competing interests

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

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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

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