Recurrent DMD Deletions Highlight Specific Role of Dp71 Isoform in Soft-Tissue Sarcomas

Abstract

Soft-tissue sarcomas (STS) are rare tumors whose oncogenesis remains unknown and for which no common therapeutic target has yet been identified. Analysis of 318 STS by CGH array evidenced a frequent deletion affecting the DMD gene (encoding dystrophin isoforms) in 16.5% of STS, including sarcomas with complex genomics, gastrointestinal tumors (GIST), and synovial sarcomas (SS). These deletions are significantly associated with metastatic progression, thus suggesting the role of DMD downregulation in the acquisition of aggressive phenotypes. We observed that targeted deletions of DMD were restricted to the 5' region of the gene, which is responsible for the transcription of Dp427. Analysis of STS tumors and cell lines by RNA sequencing revealed that only the Dp71 isoform was widely expressed. Dp427 depletion had no effect on cell growth or migration. However, Dp71 inhibition by shRNA dramatically reduced the cell proliferation and clonogenicity of three STS cell lines, likely by altering the cell cycle progression through the G2/M-phase. Our work demonstrates that DMD deletions are not restricted to myogenic tumors and could be used as a biomarker for metastatic evolution in STS. Dp71 seems to play an essential role in tumor growth, thus providing a potential target for future STS treatments.

Keywords: DMD; Dp71; cancer; myogenic sarcoma; sarcoma with complex genomics.

Figure 1

Deletion of DMD gene was significantly associated with metastatic evolution. (A) DMD expression determined by TaqMan comparing tumors with (in green) or without (in red) DMD deletion. (B) Kaplan–Meier analysis of metastasis-free survival (MFS) of sarcoma patients divided into two groups according to their DMD status (non-deleted in blue, DMD deleted in red). (C) Chromosome X (chr. X) analyzed by GCH array: losses and gains are represented in green and red, respectively. Three types of deletion affecting DMD are illustrated: deletion of entire chr. X, of the short arm of chr. X, or targeting only DMD (indicated by a red arrow). (D) Kaplan–Meier analysis of metastasis-free survival of sarcoma patients divided into two groups: (in blue) patients displaying no deletion and (in red) patients displaying either targeted DMD deletion (left plot) or deletion of p-arm/entire X chromosome (right plot). (E) Zoom on DMD genomic status on a GCH-array profile. Illustration of a typical deletion targeting the DMD gene.

Figure 2

Expression of DMD isoforms in soft-tissue sarcoma (STS) tumors and cell lines. (A) Respective expression of DMD isoforms in 145 tumors determined by RNA sequencing. Red horizontal line is minimal expression threshold: 0.001 Fragments Per Kilobase Million (FPKM) ~−9.97 log2 FPKM. (B) Expression of Dp427 and Dp71 depending on tumor histotype obtained by RNA sequencing. GIST: gastrointestinal tumor; Undiff: undifferentiated sarcoma; LMS: leiomyosarcoma; DDLPS: dedifferentiated liposarcoma; pLPS: pleomorphic liposarcoma; MFS: myxofibrosarcoma; pRMS: pleomorphic rhabdomyosarcoma; UPS: undifferentiated pleomorphic sarcoma. (C) Respective expression of Dp427 and Dp71 in sarcoma cell lines determined by RNA sequencing. (D) Respective abundance of Dp427 and Dp71 in sarcoma cell lines assessed by Western blotting.

Figure 2

Expression of DMD isoforms in soft-tissue sarcoma (STS) tumors and cell lines. (A) Respective expression of DMD isoforms in 145 tumors determined by RNA sequencing. Red horizontal line is minimal expression threshold: 0.001 Fragments Per Kilobase Million (FPKM) ~−9.97 log2 FPKM. (B) Expression of Dp427 and Dp71 depending on tumor histotype obtained by RNA sequencing. GIST: gastrointestinal tumor; Undiff: undifferentiated sarcoma; LMS: leiomyosarcoma; DDLPS: dedifferentiated liposarcoma; pLPS: pleomorphic liposarcoma; MFS: myxofibrosarcoma; pRMS: pleomorphic rhabdomyosarcoma; UPS: undifferentiated pleomorphic sarcoma. (C) Respective expression of Dp427 and Dp71 in sarcoma cell lines determined by RNA sequencing. (D) Respective abundance of Dp427 and Dp71 in sarcoma cell lines assessed by Western blotting.

Figure 3

Dp427 downregulation had no impact on cell proliferation or clonogenicity. (A) Validation by Sanger sequencing of DMD deletions induced in IB133 cell line after lentiviral transduction of the CRISPR/Cas9 system. (B) Validation by Western blot of Dp427 downregulation in cell lines deleted by the CRISPR/Cas9 system. (C) Control (in blue) or deleted (at least partially) for Dp427 (in red) cell populations were cultured for 9 days and counted by flow cytometry, with six replicates for each time point. (D) Clonogenicity was measured with crystal violet staining and plates were scanned. (E) Cells were grown to confluence in six-well plates, and then a “scratch” (wound gap) was created in the cell monolayer using a pipette tip. Images were taken at the moment of the “scratch” (0 h) and 12 h later. Results shown are representative of three independent experiments performed in duplicate.

Figure 4

Dp71 inhibition reduced cell proliferation and clonogenicity. (A) Effective Dp71 inhibition by sh1251 and sh1836 was verified by Western blotting in IB112, IB136, and SW982 cell lines incubated for 72 h without or with doxycycline. Densitometry quantification is indicated below each lane. GAPDH was used as loading control. (B) Cells were counted by flow cytometry with six replicates for each time point. Doxycycline was added (in red) in order to induce Dp71 inhibition, or not (in blue), in culture medium. (C) Clonogenicity was measured with crystal violet staining after 12 days of cell culture with (+) or without (−) doxycycline. Then, plates were scanned. (D) After 72 h incubation with or without doxycycline, cells were trypsinized, fixed, stained with propidium iodide, and analyzed by flow cytometry. Results shown are representative of three independent experiments performed in duplicate. * p-value p < 0.05, ** p < 0.01, and *** p < 0.001.