For these studies, we focused on a Drosophila
chromosomal deletion, Df(3L)vin5
, that dominantly suppresses PAX7-FOXO1–induced lethality (Supplemental Figure 2A). Human PAX7 demonstrates slightly higher sequence identity to Drosophila
PAX3/7 than does human PAX3 and was therefore used here in flies. Df(3L)vin5
deletes segments 68A2–69A1 on chromosome 3, which includes the muscle-patterning gene rols
, located at 68F1. rols
encodes an essential adaptor molecule that links the Kirre transmembrane receptor with the machinery that drives myoblast cell-cell fusion and syncytial muscle formation; therefore, rols
expression in the somatic mesoderm temporally coincides precisely with embryonic myoblast fusion (10
). However, we found by mRNA expression profiling that rols
is misexpressed in PAX7-FOXO1
larval muscle: it was reported as absent on control microarrays (n
= 3), and expressed 3.6-fold above background on PAX7-FOXO1 arrays (n
= 3) (R.L. Galindo, unpublished observations). Thus, we hypothesized that heterozygous deletion of the rols
locus might account for Df(3L)vin5
-mediated PAX7-FOXO1 suppression and that rols
might act as a PAX7-FOXO1 target gene.
Of the 2 alternative transcripts expressed from the rols
locus, only one of which is expressed in myoblasts (10
); expression of the second is restricted to endodermal/ectodermal precursors. We tested 2 rols
homozygous-lethal, P-element insertion loss-of-function alleles, P1027
, for suppression of PAX7-FOXO1. Of these 2 alleles, only the P1729
insertion disrupts expression of the myoblast rols
) (myoblast expression of rols is unperturbed in P1027
); accordingly, only the rolsP1729
allele suppressed PAX7-FOXO1–induced lethality and muscle pathogenicity (Supplemental Figure 2, A and B).
To investigate whether rols
acts as a downstream PAX-FOXO1 target, we used the daughterless-Gal4
transgene (commonly used in Drosophila
to direct strong expression of UAS-transgenes in all cells throughout development) to drive ubiquitous embryonic expression of UAS-PAX7-FOXO1
and probed for Rols misexpression. Since native Rols expression initiates at embryonic stage 11 (10
), we focused only on embryos stage 10 or earlier. Diffuse expression of PAX7-FOXO1 and Rols was observed in blastoderm (stage 4–5) embryos (Figure ), which consist of uncommitted precursor cells, and expression persisted in all examined cells — including nonmyogenic ectodermal and endodermal cells — of gastrulated (stage 9–10) embryos (Supplemental Figure 3, A–D). Taken together, these Drosophila
studies revealed that rols
acts as a PAX7-FOXO1 downstream target gene, direct or indirect, and as a bona fide genetic effector.
PAX7-FOXO1 induces Rols misexpression in Drosophila.
To extend our studies from Drosophila
to mammals, we first questioned whether rols
myoblast fusion activity is evolutionarily conserved. In mammals, 2 orthologs of rols
are present, tetratricopeptide-repeat, ankyrin-repeat, coiled-coil–containing protein 1(Tanc1
) and Tanc2
, neither of which has been studied in muscle. Since only Tanc1
is expressed in somites when myoblast fusion occurs (12
), we hypothesized that Tanc1
is the functional ortholog. We turned to mouse C2C12 cultured myoblasts, which, when switched from growth to differentiation medium (DM), differentiate and fuse to form syncytial myotubes. Quantitative RT-PCR (qRT-PCR) confirmed that Tanc1
was expressed in C2C12 cells (Figure A) and that relative expression levels decreased 40% as differentiation proceeded (Supplemental Figure 4).
Tanc1 is essential for myoblast fusion, but not for myogenic differentiation.
We next used shRNAs to establish that Tanc1 activity is essential for myotube formation. We tested 2 separate constructs, A6 and A10, individually and in combination and used qRT-PCR to confirm mRNA knockdown (Figure A). Tanc1 silencing potently blocked the formation of syncytial myotubes in a dose-dependent manner (Figure , B, D, and E). Tanc1-silenced cells, however, still transitioned from round precursors to spindle-shaped cells (Figure , D and E), suggestive of successful myocyte differentiation. Immunofluorescence for muscle-specific myosin heavy chain (MHC; a marker of terminal differentiation) confirmed that Tanc1 shRNA–treated myoblasts successfully switched to a differentiated state (Figure , C and E). Thus, the mechanisms that drive cell-cell fusion uncouple from the myogenic signals that induce myocyte terminal differentiation.
Similar to the rols/PAX7-FOXO1 genetic interaction in Drosophila, Tanc1 was critical for PAX-FOXO1 pathogenicity in mammalian myoblasts. We used retroviral-mediated gene transfer to generate stable PAX3-FOXO1–expressing C2C12 myoblasts, as PAX3-FOXO1 is the more common RMS chimera and the form most often profiled in mammalian cells, and confirmed that PAX3-FOXO1 protein misexpression levels were comparable to human PAX3-FOXO1 RMS cultured cells (specifically, RMS-13 cells; Supplemental Figure 5; also shown is Drosophila larval PAX7-FOXO1 expression). We found that PAX3-FOXO1 induced overexpression of Tanc1 (Figure A) and that, like RMS cells, PAX3-FOXO1 myoblasts demonstrated neoplasia-related phenotypes, including impaired myogenic differentiation and an inability to form myotubes (Figure , B and C, and Supplemental Figure 6A). However, reducing Tanc1 expression back to normal levels markedly suppressed PAX3-FOXO1 pathogenicity, as transient transfection of Tanc1 shRNA into PAX3-FOXO1 myoblasts restored both differentiation and fusion potential (Figure , B and C). It is worth noting that in PAX3-FOXO1 cells, the A6 hairpin alone demonstrated the most effective rescue. Immunoblot analysis confirmed that PAX3-FOXO1 protein levels remained unchanged (Supplemental Figure 6B). Thus, Tanc1 is a critical PAX3-FOXO1 downstream effector.
TANC1 silencing rescues differentiation arrest and failed fusion of PAX3-FOXO1 cells and marks RMS tumor cells.
To determine whether Tanc1 overexpression itself perturbs myoblast fusion and/or differentiation, we again used retroviral-mediated gene transfer to generate C2C12 cells that constitutively overexpress Tanc1 (Supplemental Figure 7A). Like PAX3-FOXO1 cells, Tanc1-infected myoblasts were severely crippled with regard to myoblast fusion potential; however, unlike PAX3-FOXO1 cells, Tanc1 myoblasts differentiated normally into myocytes (Supplemental Figure 7B). These finding again show that fusion potential can be uncoupled from myocyte terminal differentiation.
We next turned to RMS-13 cells to establish that human TANC1 influences RMS. RMS-13 cells, similar to RMS myoblasts in vivo, exhibited little to no expression of MHC or fusion potential (Figure , E and F, and Supplemental Figure 8A). Treatment of RMS-13 cells with shRNA — the A6 and A10 hairpins, which target both mouse Tanc1 and human TANC1 — again reduced the relative expression of TANC1 mRNA as well as TANC1 protein steady-state levels, whereas PAX3-FOXO1 protein levels remained unchanged (Figure D and Supplemental Figure 8, A and B). When switched to DM, TANC1-silenced RMS-13 myoblasts transitioned from polygonal myoblasts to spindled cells (Supplemental Figure 8C), suggestive of differentiation into myocytes. Immunofluorescence confirmed that TANC1-silenced RMS-13 cells differentiated and fused to form MHC-positive syncytia (Figure , E and F). Consistent with these findings, TANC1-silenced RMS-13 cells showed markedly diminished oncogenicity, as demonstrated by decreased anchorage-independent growth and colony formation on soft agar (Supplemental Figure 8D).
Finally, immunohistochemistry showed that TANC1 protein was strongly expressed in PAX3-FOXO1 RMS tumors (n = 5; Figure H) compared with control childhood skeletal muscle tissue (n = 3; Figure G). These findings highlight the notion that TANC1 misexpression can be used to mark PAX-FOXO1 RMS tumor cells.
RMS model systems conveniently promote insights into not only neoplasia, but also muscle development. Although ultrastructural studies suggest that myoblast fusion biology is conserved (13
), few of the Drosophila
fusigenic genes have been identified as essential in mammals (13
), and none of these were from the founder subfamily. As the name implies, founder myoblasts are seminal to Drosophila
myogenesis, uniquely dictating the location and physiology of each individual muscle (21
). With rols
, we have now shown that founder gene function is conserved in mammals and, furthermore, participates in human disease. How founder gene activity influences other forms of neuromuscular disease now becomes an intriguing issue.
Regarding RMS, we conclude that: (a) Tanc1 is essential for mammalian myoblast fusion, but is dispensable for the myogenic differentiation of wild-type cells; (b) PAX-FOXO1 signaling drives Tanc1 overexpression; (c) reducing rols/Tanc1/TANC1 activity suppresses gain-of-function PAX-FOXO1 pathogenicity in multiple independent model systems, highlighting TANC1 as a critical PAX-FOXO1 downstream effector; and (d) TANC1 activity and altered myoblast fusion mechanistically contribute to PAX-FOXO1 RMS.
Genetic screening in a Drosophila
model and loss-of-function/gain-of-function studies in mammalian platforms have collaboratively uncovered a PAX-FOXO1→TANC1 neoplasia axis, a finding we believe to be novel. Our results also argue that the relationship between myogenesis transcription factor (e.g., MyoD) signaling and myoblast fusion genes is intricate. In the presence of altered fusion potential, both Drosophila
) and mammalian myoblasts (present study) transition to differentiated myocytes, which suggests that later aspects of myogenesis signaling must uncouple from the TANC1
fusigenic pathway. Yet correcting PAX-FOXO1–mediated overexpression of rols
rescued PAX-FOXO1–induced differentiation and arrest. These results intimate that correction of the TANC1 fusigenic axis feeds back to and rescues PAX-FOXO1–mediated misregulation of myogenic signaling, raising fascinating questions regarding the mechanisms by which this occurs. Our observation that PAX3-FOXO1 protein levels remained unchanged in TANC1
-silenced cells argues that rescue does not originate from decreased expression of PAX3-FOXO1 from the PAX3 promoter. Thus, we speculate that rescue occurs epistatically downstream of PAX3-FOXO1.
Interestingly, myoblasts were remarkably sensitive to modest variations in TANC1 expression levels. A relatively wide range of rescue penetrance was observed for the 2 TANC1 shRNA hairpins and combined cocktail, even though the relative expression levels of mouse Tanc1 or human TANC1, when treated by the various shRNAs, differed by 38% and 35%, respectively. We note, however, that the relative levels of Tanc1 gene expression decreased only 40% over the normal course of differentiation. Thus, we favor the notion that a relatively precise requirement for appropriate TANC1 expression is biologically relevant for both myoblast fusion and RMS pathobiology.
Reprogramming neoplastic precursor cells to undergo terminal differentiation — commonly referred to as differentiation therapy — is of particular clinical interest, as it is often substantially less toxic than general chemotherapeutic agents (e.g., use of retinoic acid in acute promyelocytic leukemia). Conceptually, targeting the myoblast fusion pathway may represent a new avenue for PAX-FOXO1 RMS differentiation therapy, although whether an equivalent TANC1 axis participates in PAX-FOXO1–negative (i.e., embryonal) RMS remains a provocative question. Of note, Yang and colleagues have demonstrated that forced inhibition of MyoD in embryonal RMS cells prompts terminal differentiation (22
). Therefore, RMS in general appears to be a clinically ripe candidate for differentiation therapy.