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. 2010 Jan;176(1):416-34.
doi: 10.2353/ajpath.2010.090405. Epub 2009 Dec 17.

Mice Lacking Dystrophin or Alpha Sarcoglycan Spontaneously Develop Embryonal Rhabdomyosarcoma With Cancer-Associated p53 Mutations and Alternatively Spliced or Mutant Mdm2 Transcripts

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Free PMC article

Mice Lacking Dystrophin or Alpha Sarcoglycan Spontaneously Develop Embryonal Rhabdomyosarcoma With Cancer-Associated p53 Mutations and Alternatively Spliced or Mutant Mdm2 Transcripts

Karen Fernandez et al. Am J Pathol. .
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Abstract

Altered expression of proteins in the dystrophin-associated glycoprotein complex results in muscular dystrophy and has more recently been implicated in a number of forms of cancer. Here we show that loss of either of two members of this complex, dystrophin in mdx mice or alpha sarcoglycan in Sgca(-/-) mice, results in the spontaneous development of muscle-derived embryonal rhabdomyosarcoma (RMS) after 1 year of age. Many mdx and Sgca(-/-) tumors showed increased expression of insulin-like growth factor 2, retinoblastoma protein, and phosphorylated Akt and decreased expression of phosphatase and tensin homolog gene, much as is found in a human RMS. Further, all mdx and Sgca(-/-) RMS analyzed had increased expression of p53 and murine double minute (mdm)2 protein and contained missense p53 mutations previously identified in human cancers. The mdx RMS also contained missense mutations in Mdm2 or alternatively spliced Mdm2 transcripts that lacked an exon encoding a portion of the p53-binding domain. No Pax3:Fkhr or Pax7:Fkhr translocation mRNA products were evident in any tumor. Expression of natively glycosylated alpha dystroglycan and alpha sarcoglycan was reduced in mdx RMS, whereas dystrophin expression was absent in almost all human RMS, both for embryonal and alveolar RMS subtypes. These studies show that absence of members of the dystrophin-associated glycoprotein complex constitutes a permissive environment for spontaneous development of embryonal RMS associated with mutation of p53 and mutation or altered splicing of Mdm2.

Figures

Figure 1
Figure 1
mdx and Sgca−/− mice develop skeletal muscle-derived tumors with age. Mice lacking dystrophin (mdx) or α sarcoglycan (Sgca−/−) developed skeletal muscle-derived tumors, primarily from the axial musculature, after 12 months of age. Examples of tumors emanating from periorbital muscles (A), muscles in the hindlimb/buttocks (B, C), or forelimb muscles (D, E) are shown.
Figure 2
Figure 2
mdx and Sgca−/− tumors are embryonal rhabdomyosarcoma. Tumors were snap-frozen, cut, and stained with H&E. All tumors showed characteristics of embryonal rhabdomyosarcoma, including high cellularity (A–C, F) with evidence of muscle differentiation (B). A small fraction of tumors showed anaplasia (D) and a few contained very small areas (ca. 1% of total) with an alveolar-like pattern (E). RMS cells were present within regions bounded by and containing dystrophic skeletal myofibers (G) and often contained rhabdomyoblasts (H). Scale bars: 200 μm (A–C), 100 μm (D–F), and 50 μm (G–H).
Figure 3
Figure 3
Expression of RMS markers in mdx and Sgca−/− tumors. Tumor sections were stained with antibody to MyoD, myogenin, desmin, or with secondary antibody alone. All sections were counterstained with hematoxylin. Every mdx and Sgca−/− tumor contained tumor cells expressing MyoD, myogenin, and desmin. Scale bar: 100 μm (left panels) or 50 μm (right panels).
Figure 4
Figure 4
Expression of cancer-related proteins in mdx RMS and normal mdx and wild-type (WT) skeletal muscle. Forty micrograms of whole muscle protein lysate from wild-type skeletal muscle (WT), mdx skeletal muscle (mdx), or mdx RMS tumor (mdx RMS) were separated on SDS-PAGE gels and blotted with the indicated antibodies. All mdx RMS overexpressed p53 and Mdm2, while some, but not all, mdx RMS overexpressed Igf2, phospho-serine473-Akt, survivin, and Rb or had reduced levels of PTEN. Native molecular weights, indicated by asterisks when multiple bands were present, are: Pax3/7 (56 kDa), Fkhr (80 kDa), p53 (53 kDa) Rb (106 kDa), Mdm2 (90 kDa), pre-pro IGF1 (24 kDa), pre-pro IGF2 (18 kDa), Akt (60 kDa), PTEN (54 kDa), survivin (17 kDa), desmin (53 kDa), and actin (43 kDa).
Figure 5
Figure 5
mRNA levels for Pax3/7, Fkhr, and possible Pax3/7-Fkhr translocation products in mdx RMS. mRNA for Pax3 and Pax7 (cumulative, Pax3/7), Fkhr, and potential 5′ Pax3/7–3′Fkhr translocation products were assessed by endpoint RT-PCR, compared with βactin control, in mdx RMS tumors and wild-type (WT) and mdx skeletal muscle. C is control with no template added. Asterisks indicate the expected molecular weight of full-length amplified cDNA.
Figure 6
Figure 6
Expression of cell death proteins in mdx RMS and normal mdx and wild-type skeletal muscle. Forty micrograms of whole muscle protein lysate from mdx RMS and mdx and wild-type (WT) skeletal muscle were separated by SDS-PAGE and immunoblotted to show relative expression of proteins involved in the regulation of apoptosis. Most mdx RMS had increased expression of pre-pro caspase 3 (but not 12 kDa or 17 kDa activated forms), BID (a pro-apoptosis factor), and reduced levels of GAPDH (an apoptosis inhibitor). Bax and Bcl-2 were elevated in mdx muscle versus wild-type, but not increased further in mdx RMS. Native molecular weights are: apoptosis inhibiting factor (65 kDa), Bax (23 kDa), Bcl-2 (26 kDa), BID (22 kDa), GAPDH (36 kDa), pre-pro caspase 3 (36/33 kDa), pre-pro caspase 9 (46 kDa), and actin (43 kDa).
Figure 7
Figure 7
Reduced expression of glycosylated α dystroglycan and α sarcoglycan in mdx RMS. Forty micrograms of total cell protein was compared for expression of glycosylated α dystroglycan (using IIH6), β dystroglycan, or α sarcoglycan. Glycosylated α dystroglycan expression and α sarcoglycan protein expression were reduced in about half of mdx RMS, while β dystroglycan was not. Asterisks indicate the molecular weight of native full-length protein. Native molecular weights are: α dystroglycan (160 kDa), β dystroglycan (43 kDa), α sarcoglycan (50 kDa).
Figure 8
Figure 8
α Dystroglycan, and α and β sarcoglycan immunostaining are reduced in mdx RMS. Tumor sections were immunostained with antibodies to dystroglycans or sarcoglycans. Variably decreased expression of α dystroglycan (IIH6) and α and β sarcoglycan were found in mdx RMS, while β dystroglycan was always strongly expressed. Scale bar = 100 μm.
Figure 9
Figure 9
Expression of dystrophin and α sarcoglycan are reduced in human RMS. Forty micrograms of whole cell protein was extracted from normal pediatric human muscle (normal) or from pediatric human RMS (subtype unspecified), separated by SDS-PAGE, and blotted for dystrophin, α sarcoglycan, desmin, or actin. Asterisks indicate molecular weight of native full-length protein: dystrophin (427 kDa), α sarcoglycan (50 kDa), desmin (53 kDa), and actin (43 kDa).
Figure 10
Figure 10
Dystrophin is not expressed in most pediatric RMS, regardless of subtype. Tissue arrays bearing sections of pediatric normal skeletal muscle (SM), embryonal rhabdomyosarcoma (ERMS), or alveolar rhabdomyosarcoma (ARMS) were stained with Dys1, an antibody to dystrophin. All SM stained strongly along the sarcolemmal membranes of myofibers, the normal location of dystrophin protein, whereas almost no RMS, either ERMS or ARMS, were stained. When RMS did express dystrophin, it was present only in a small minority of the tumor cells within the section. Skeletal myofibers within RMS sections (arrowheads), however, stained strongly for dystrophin. Scale bar = 100 μm.

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