Cancer Cell, Volume 26 Supplemental Information The Hippo Transducer YAP1 Transforms Activated Satellite Cells and Is a Potent Effector of Embryonal Rhabdomyosarcoma Formation Annie M. Tremblay, Edoardo Missiaglia, Giorgio G. Galli, Simone Hettmer, Roby Urcia, Matteo Carrara, Robert N. Judson, Khin Thway, Gema Nadal, Joanna L. Selfe, Graeme Murray, Raffaele A. Calogero, Cosimo De Bari, Peter S. Zammit, Mauro Delorenzi, Amy J. Wagers, Janet Shipley, Henning Wackerhage, and Fernando D. Camargo
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Supplemental Information The Hippo Transducer YAP1 ... · Activated Satellite Cells and Is a Potent Effector . of Embryonal Rhabdomyosarcoma Formation . Annie M. Tremblay, ... Henning
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Cancer Cell, Volume 26
Supplemental Information
The Hippo Transducer YAP1 Transforms
Activated Satellite Cells and Is a Potent Effector
of Embryonal Rhabdomyosarcoma Formation
Annie M. Tremblay, Edoardo Missiaglia, Giorgio G. Galli, Simone Hettmer, Roby Urcia, Matteo Carrara, Robert N. Judson, Khin Thway, Gema Nadal, Joanna L. Selfe, Graeme Murray, Raffaele A. Calogero, Cosimo De Bari, Peter S. Zammit, Mauro Delorenzi, Amy J. Wagers, Janet Shipley, Henning Wackerhage, and Fernando D. Camargo
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SUPPLEMENTAL DATA
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Figure S1, related to Figure 2. (A) Images showing the hindlimbs flexibility from control (left) and induced Myf5Cre-hYAP1 S127A mice (right). (B) x-ray imaging showing excised limbs from control (left) and induced Myf5Cre-hYAP1 S127A mice (right). (C-D) Cross-section of the brown fat pads of adult control (C) and induced Myf5Cre-hYAP1 S127A mice (D) stained by H&E. Scale bars, 50 μm. (E) Diagram of the allograft transplantation studies with Myf5Cre-hYAP1 S127A donors. (F-I) Cross-section of the allografts tumors from (E), stained by H&E (F) and immunostained for YAP1 (G), Desmin (H) and Myogenin (I). Scale bars, 50 μm. (J) Diagram of the secondary transplantation experiments. (K) Relative latency per number of cells transplanted. (n=2/dilution) (L-M) Primary tumors of the TA in Myod1-hYAP1 S127A mice after 4 weeks of DOX induction. (N-O) Images of tumors in (M), stained by H&E. Scale bars, 200 µm (N) and 100 um (O). Yellow arrowheads mark centrally located nuclei. (P) Diagram of the allograft transplantation studies with Myod1-iCre-hYAP1 S127A donors. (Q-) Cross-section of the allografts tumors from (P), stained by H&E (Q) and immunostained for YAP1 (R), Desmin (S) and Myogenin (T). Scale bars, 50 μm (Q, R) and 100 μm (S, T). (U) C2C12 myoblasts infected with a hYAP1 S127A retroviral expression virus or control virus, grown in soft agar. Quantification was performed after 15 days in culture. n=4, p <0.001. Scale bar, 500 μm.
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Figure S2, related to Figure 3. (A) As in (Fig 3C), immunostained for PAX7. (B) Quantification of PAX7 immunostaining as in (A). Results are presented as fold change of the percentage of PAX7+ nuclei/total nuclei/10X field of view (FOV) +/- SD. (6 FOV/condition analyzed, total of counted nuclei are 870 for control and 920 for Pax7-YAP1 S127A mice). (C-G) As in (Fig 3D), immunostained for YAP1 (C), PCNA (D), Desmin (E), Myogenin (F) and PAX7 (G). All scale bars, 100 μm
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Figure S3, related to Figure 4. (A-B) Diagram of the ZsGreen1-traced YAP1 -ERMS tumor cells generation and culture (A) and the resulting tumors following transplantation/regression of ZsGreen+ YAP1 -ERMS cells (B). Dashed line circumscribes the tumor location. Scale bars, 1 cm. (C) EdU incorporation in cultured ZsGreen1-traced tumor cells in the presence and absence of DOX. (n=3). Scale bars, 50 μm. (D) Quantification of EdU incorporation as in (C) (presented as mean +/- SEM). (E) Immunofluorescence staining for MyHC in differentiated ZsGreen1-traced tumor cells in the presence and absence of DOX (n=3). Scale bars, 50 μm.
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Figure S4, related to Figure 5. (A) Fold Change of hYAP1, Cyr61, Myf5, Pax7, Myod1 and Myh4 mRNA levels by qPCR in control muscle and during tumor regression after DOX withdrawal for 0 (TUM), 3 (OFF3) and 6 days (OFF6) (n=3; Fold change +/- SEM). (B) Diagram from Ingenuity Pathway Analysis (IPA) of the 249 downregulated genes in the YAP1 -ERMS tumors versus control muscle. Table S1, related to Figure 5. Provided as an Excel file.
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Figure S5, related to Figure 6. (A) Enrichment of Myogenin, MEF2 and SRF binding sites within the TEAD1 peaks identified by ChIP-Seq in YAP1 -ERMS cells. Analysis from GREAT annotation. (B) Heat map of the TEAD1-bound sites in YAP1 -ERMS clustered according to the presence of shared loci with MYOD1, Myogenin and SRF in C2C12 myotubes. (C) Gene ontology analysis of the shared TEAD1/MYOD1/Myogenin/SRF binding sites. (D) Distribution of the expression change of genes from the Malignant Muscle Neoplasm and the Muscle Contraction categories in (C). (E) Gene ontology analysis of the TEAD1 only sites. (F) Heat map of the TEAD1-bound sites in YAP1 -ERMS clustered according to the presence of shared loci with MYOD1 and Myogenin in C2C12 myoblasts and myotubes. (G-J) Target genes validated by ChIP-qPCR for YAP1 (G), TEAD1 (H), MEF2 (I) and MYOD1 (J) occupancy in RD cells. Fold enrichment over IgG normalized to 1 (red line) +/- SD. Negative region is -8.3 kb upstream of CTGF TSS. (K) Heat map showing expression of YAP1 /TEAD1 validated target genes in RD cells versus skeletal muscle control (SKM). (L) Heat map showing expression of YAP1 /TEAD1 validated target genes in human ERMS tumors versus skeletal muscle control (SKM). (M-O) Expression score of the YAP1 gene (M), YAP1-ERMS-activated signature (N) and YAP1-ERMS-repressed signature (O) expression scores in human fetal myoblasts undergoing differentiation (public dataset GSE3780).
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Figure S6, related to figure 7. (A-B) Radar plot showing the level of enrichment for YAP1-ERMS-activated signature and other relevant signal transduction pathways in human RMS versus skeletal muscle (A) and in ERMS versus ARMSp (B). The - log10 of the p value of the hypergeometric test for each signature was reported in a radar plot (p value cupped at 0.0001). Grey area represents p values below significance after Bonferroni correction.
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Table S2, related to Figure 7. Provided as an Excel file. Sonic hedgehog homolog (SHH) signature: Shi et al., 2010 Embryonic Stem Cell (ES) signature: Ben-Porath et al., 2008. Activated satellite cells (Satell−act.) signature: Pallafachina et al., 2010. β-Catenin signature: Bild et al., 2006. ERBB2 signature: Mackay et al., 2003. MYC signature: Bild et al., 2006. NOTCH signature: Mazzone et al., 2010. RAS signature: Bild et al., 2006. SRC signature: Bild et al., 2006. TGF-β signature: Padua et al., 2008. STAT3 signature: Alvarez et al., 2005. WNT signature: Biocarta (http://www.broadinstitute.org/gsea/msigdb/cards/BIOCARTA_WNT_PATHWAY). Table S3, related to Figure 7.
Variables Coefficients CI 95% p Value (Intercept) -0.18 (-0.58 / 0.22) 0.374 Histology (baseline: Fusion Negative RMS)
Multivariable linear regression modeling the relationship between YAP1 -ERMS_activated score and other clinico-pathological variables such as fusion status, tumor stage, patient age and tumor location. * These variables were categorized as previously reported (Missiaglia et al, 2012). Model F-statistic: 3.395 on 10 and 223 DF, p = 0.00038.
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Mackay A, Jones C, Dexter T, Silva RL, Bulmer K, Jones A, Simpson P, Harris RA, Jat PS, Neville AM, Reis LF, Lakhani SR and O'Hare MJ. (2003) cDNA microarray analysis of genes associated with ERBB2 (HER2/neu) overexpression in human mammary luminal epithelial cells. Oncogene 22 , 2680-8 Mazzone M, Selfors LM, Albeck J, Overholtzer M, Sale S, Carroll DL, Pandya D, Lu Y, Mills GB, Aster JC, Artavanis-Tsakonas S and Brugge JS. (2010) Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells. PNAS 107 , 5012-7 McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, Wenger AM, and Bejerano G. (2010) GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol 28 , 495-501. Padua D, Zhang XH, Wang Q, Nadal C, Gerald WL, Gomis RR, and Massagué J. (2008) TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell 133, 66-77 Shi T, Mazumdar T, Devecchio J, Duan ZH, Agyeman A, Aziz M and Houghton JA. (2010) cDNA microarray gene expression profiling of hedgehog signaling pathway inhibition in human colon cancer cells. PLoS One 5 , pii: e13054 Smyth GK, Michaud J and Scott HS. (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics. 21 , 2067-75 Ye T, Krebs AR, Choukrallah MA, Keime C, Plewniak F, Davidson I and Tora L. (2011) seqMINER: an integrated ChIP-seq data interpretation platform. Nucleic Acids Res. 39 , e35 Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W and Liu XS. (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9 , R137