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Dystrophin is a tumor suppressor in human cancers with myogenic programs

Abstract

Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation1,2,3. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy–associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer.

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Figure 1: Identification of somatic intragenic DMD deletions in human myogenic cancers.
Figure 2: MLPA evaluation of DMD exons 1–79 shows intragenic deletions in 24 myogenic cancers.
Figure 3: Loss of expression of 427-kDa dystrophin in most metastatic GIST, RMS and LMS tumors.
Figure 4: Expression of Dp71 dystrophin in myogenic tumors.
Figure 5: Restoration of dystrophin expression inhibits invasiveness and migration in DMD-inactivated GIST, RMS and LMS cells but not in a comparator non-myogenic fibrosarcoma cell line (HT-1080).
Figure 6: Restoration of dystrophin expression inhibits anchorage-independent growth and invadopodia formation in DMD-inactivated myogenic cancer cells.

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Acknowledgements

We thank J. Tremblay (Quebec University Hospital) for the pCR3.1-miniDMD construct; T. Taguchi (Kochi Medical School) for the GIST-T1 cell line; Y. Hayashi, M. Bardsley and H. Qiu for their expert technical assistance; and S. Bauer (West German Cancer Center) for useful discussions and for funding support of J.H. This work was supported by grants from the US National Institutes of Health, including 1P50CA127003-07 (J.A.F. and G.D.D.), 1P50CA168512-01 (J.A.F., G.D.D. and A.M.-E.) and 5R01DK058185-12 (T.O.), and by the Virginia and Daniel K. Ludwig Trust for Cancer Research (J.A.F. and G.D.D.), the GIST Cancer Research Fund (J.A.F.), the Life Raft Group (J.A.F., T.O. and M.v.d.R.), the Cesarini Pan-Mass Challenge for GIST (J.A.F. and G.D.D.), Paul's Posse of the Pan-Mass Challenge (J.A.F. and G.D.D.), the Bernard F. and Alva B. Gimbel Foundation (L.M.K.), the Sarcoma Alliance for Research Through Collaboration (A.M.-E.) and the Erica Kaitz LMS Research NOW Fund (J.A.F., A.M.-E. and G.D.D.).

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Contributions

J.A.F. supervised the project. Y.W. and J.A.F. generated the original hypothesis and designed the study. Y.W., A.M.-E., R.R.B., M.Z., Y.S., G.E., J.-C.L., J.H., B.S.F., Z.G., Y.S., X.G. and T.O. performed experiments. A.M.-E., C.R.A., C.D.M.F., C.P.R., M.v.d.R. and J.A.F. provided samples and clinical data. Y.W., A.M.-E., R.R.B., M.Z., Y.S., G.E., J.-C.L., B.S.F., Z.G., E.A.F., X.G., M.v.d.R., T.O., L.M.K. and J.A.F. analyzed data. C.R.A., C.D.M.F., G.D.D., M.v.d.R. and L.M.K. provided scientific advice and helpful comments on the project. Y.W., A.M.-E., G.E. and J.A.F. wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jonathan A Fletcher.

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Integrated supplementary information

Supplementary Figure 1 Identical DMD deletions in a primary GIST and subsequent metastasis.

Identical DMD deletions in a primary gastric GIST and a subsequent metastasis, diagnosed 1 year later, from patient 31. SNP profiles of non-neoplastic DNA from the patient, the primary gastric GIST and the subsequent metastasis are shown. The top panel (a) shows the entire chromosome X; the bottom panel (b) focuses on the DMD locus. Data are shown as dChip SNP log2 ratio copy number.

Supplementary Figure 2 Identical DMD deletions in multiple GIST metastases from one patient.

Identical DMD deletions in each of 28 GIST metastases from patient 61. The metastases have a DMD deletion not present in non-neoplastic DNA from the patient, demonstrating the somatic nature of DMD deletion. The top panel (a) shows the entire chromosome X; the bottom panel (b) focuses on the DMD locus. Data are shown as dChip SNP log2 ratio copy number, with a representative SNP profile (metastasis 10) shown to the right.

Supplementary Figure 3 DMD intragenic deletions in 1,003 human cancers.

DMD intragenic deletions in 1,003 human cancers, including 40 myogenic sarcomas, 58 non-myogenic sarcomas and 905 non-sarcoma cancers. The 58 non-myogenic sarcomas (Supplementary Table 2) include 4 malignant peripheral nerve sheath tumors, 10 liposarcomas, 15 Ewing's sarcomas, 1 fibrosarcoma, 1 clear cell sarcoma, 1 spindle cell sarcoma, 1 dermatofibrosarcoma protuberans, 1 giant cell tumor, 1 mesenchymal chondrosarcoma and 23 osteosarcomas. The 905 non-sarcoma cancers (Cancer Cell Line Encyclopedia; Barretina et al. Nature, 2012) include 591 carcinomas, 175 hematologic neoplasms, 59 melanomas, 43 gliomas, 15 neuroblastomas and 22 other tumors. DMD intragenic deletions are significantly more frequent in myogenic sarcomas than in non-myogenic sarcomas (P < 0.0001, two-tailed Fisher's test) and non-sarcoma cancers (P < 0.0001, two-tailed Fisher's test).

Supplementary Figure 4 Detailed characterization of intragenic DMD deletions by MLPA.

DMD MLPA capillary electropherograms are normal in low-risk GISTs from male (a) and female (b) patients. MLPA shows intragenic hemizygous, homozygous and heterozygous deletions in metastatic GISTs from a male (c) and two female (d,e) patients, respectively. Each peak represents a single DMD exon, displayed non-consecutively on the basis of the size of the ligated stuffer sequence. The reference trace is shown in red, and the tumor sample is shown in blue.

Supplementary Figure 5 Expression of Dp71 dystrophin in non-myogenic sarcomas.

Protein blotting with dystrophin antibody 7A10 demonstrates Dp71 expression in malignant peripheral nerve sheath tumor (MPNST), liposarcoma (LPS), dermatofibrosarcoma protuberans (DFSP) and malignant solitary fibrous tumor (SFT).

Supplementary Figure 6 Dp71 dystrophin is a positive regulator of cell viability in myogenic sarcoma.

(a) Protein blot validation of siRNA-mediated inhibition of Dp71 in RMS176 (eRMS with DMD deletion) and RMS843 (aRMS without DMD deletion). (b) Dp71 inhibition reduces cell viability, as assessed by CellTiter-Glo assay. The control is a non-targeting siRNA. Data are shown as the mean ± s.d. from three replicates. Dp71 and Dp427 were detected with dystrophin antibodies 7A10 and DYS1, respectively.

Supplementary Figure 7 miniDMD expression in GIST, RMS and LMS cells at levels physiological for dystrophin in the corresponding cell lineages.

Stable transfection with miniDMD of GIST, RMS and LMS cells induces expression of 240-kDa miniDMD dystrophin at levels comparable to those of endogeneous 427-kDa dystrophin in low-risk GIST, skeletal muscle and myometrium cells, respectively. miniDMD was detected with DYS2 antibody.

Supplementary Figure 8 Effect of miniDMD expression on cell viability in DMD-inactivated GIST, RMS and LMS cells.

Stable transfection with miniDMD reduces cell viability in RMS176 and LMS04 cells, as assessed by CellTiter-Glo viability assay, but does not alter the viability of GIST (GIST-T1 and GIST430) or non-myogenic cells: fibrosarcoma (HT-1080), Ewing's sarcoma (EWS502) and HEK 293. Data are shown as the mean ± s.d. of three replicates.

Supplementary Figure 9 Dystrophin is not expressed in Ewing's sarcoma (EWS502 and EWS894), fibrosarcoma (HT-1080) and HEK 293 cells.

Dystrophin was detected with DYS1 antibody.

Supplementary Figure 10 Dystrophin immunohistochemistry in clinical samples.

Immunohistochemical detection in formalin-fixed, paraffin-embedded surgical samples shows robust dystrophin expression in skeletal, cardiac and smooth muscle (a) and in benign smooth muscle neoplasms (b, top), whereas dystrophin expression is inhibited in malignant smooth muscle tumors (b, bottom; LMS, leiomyosarcoma). (c) Contingency table summarizing dystrophin expression assessed by immunohistochemistry in 20 benign and 57 malignant smooth muscle tumors in a tissue microarray. Loss of dystrophin expression correlates with malignancy in smooth muscle tumors (P < 0.0001, two-tailed Fisher's test).

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Wang, Y., Marino-Enriquez, A., Bennett, R. et al. Dystrophin is a tumor suppressor in human cancers with myogenic programs. Nat Genet 46, 601–606 (2014). https://doi.org/10.1038/ng.2974

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