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Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

  1. Inês M Tenente
  2. Madeline N Hayes
  3. Myron S Ignatius
  4. Karin McCarthy
  5. Marielle Yohe
  6. Sivasish Sindiri
  7. Berkley Gryder
  8. Mariana L Oliveira
  9. Ashwin Ramakrishnan
  10. Qin Tang
  11. Eleanor Y Chen
  12. G Petur Nielsen
  13. Javed Khan
  14. David M Langenau  Is a corresponding author
  1. Cancer Center, and Regenerative Medicine, Massachusetts General Hospital, United States
  2. Harvard Stem Cell Institute, United States
  3. Abel Salazar Biomedical Sciences Institute, University of Porto, Portugal
  4. Greehey Children's Cancer Research Institute, United States
  5. Pediatric Oncology Branch, Advanced Technology Center, National Cancer Institute, United States
  6. Faculdade de Medicina, Universidade de Lisboa, Portugal
  7. University of Washington, United States
  8. Massachusetts General Hospital, United States
Research Article
Cite this article as: eLife 2017;6:e19214 doi: 10.7554/eLife.19214
6 figures, 1 table and 4 additional files

Figures

Figure 1 with 3 supplements
Transgenic myf5 elevates tumor cell differentiation, increases tumor size, and accelerates time to primary tumor-onset when expressed in myosin-expressing ERMS cells.

(AF) Primary ERMS developing in myf5:GFP/mylpfa:mCherry AB-strain zebrafish. Transgenic kRASG12D-expressing ERMS (AC) compared with those that express both kRASG12Dand mylpfa:myf5 (DF). Animals imaged at 35 dpf (A,D). Hematoxylin and Eosin-stained sections of representative tumors (B,E) and quantification of differentiation within individual tumors (C,F; 1-less differentiated and 3-most differentiated). Asterisk denotes p=0.015 by Chi-square test. (G) Quantitative real-time PCR gene expression performed on bulk ERMS cells, confirming high myf5 expression, increased differentiation, and high expression of TPC associated genes in ERMS that co-express kRASG12Dand mylpfa:myf5 (K+M, N = 5). Endogenous myf5 was assessed using primers specific to the 3’UTR and total myf5 assessed by primers that amplify the coding sequence (cds). cadherin 15 (cdh15) and myogenin (myog). kRASG12D alone expressing ERMS (K, N = 4). Average gene expression with 50% confidence intervals denoted by box. Mean, maximum, and minimum also denoted. (H) Relative tumor size of primary ERMS at 30 days post fertilization (dpf). Box shows 50% confidence interval. Mean, maximum, and minimum denoted. Asterisk denotes p=0.0108, Student’s t-test. (I) Kaplan-Meijer analysis denoting time-to-tumor onset (p<0.001, Log-rank Statistic, N = 494 fish analyzed for K and N = 470 for K+M). Scale bars equal 2 mm (A,D) and 50 μm (B,E). Asterisks in panels G-H denote *p<0.05; **p<0.01; ***p<0.001 by Student’s t-test.

https://doi.org/10.7554/eLife.19214.002
Figure 1—figure supplement 1
Fluorescence images of primary ERMS developing in stable transgenic myf5:GFP/mylpfa:mCherry zebrafish.

Images of the same representative rag2:kRASG12D –alone (AC) and rag2:kRASG12D; mylpfa:myf5 (DF) zebrafish shown in Figure 1A and D, respectively. (A,D) merged (brightfield, GFP and mCherry) image. (B,E) mCherry image. (C,F) GFP image. Scale bars equal 2 mm.

https://doi.org/10.7554/eLife.19214.003
Figure 1—figure supplement 2
Histological classification of primary zebrafish ERMS based on differentiation score.

Representative H and E-stained sections of zebrafish ERMS assigned to each differentiation category. Scale bars equal 100 μm.

https://doi.org/10.7554/eLife.19214.004
Figure 1—figure supplement 3
Analysis of proliferation and apoptosis in zebrafish primary ERMS.

(A) Representative H and E-stained sections and immunohistochemistry for phospho-H3 (pH3) and cleaved caspase-3 (CC3). (B) Quantification of the total number of pH3-positive cells per 400x imaging field. (n=average of 3 fields/tumor section). (C) Quantification of the total number of CC3-positive cells per 400x imaging field (n=average of 3 fields/tumor). Boxes in BC denote 50% confidence interval and mean, maximum, and minimum shown. kRASG12D[K] (N = 5) and kRASG12D; mylpfa:myf5 [K+M] (N = 11). (D) Quantification of total number of EdU+ cells per area (n=average of 3 fields/tumor. N = 3 tumors per genotype). *p<0.05 or **p<0.01 in comparison to each kRASG12D-alone expressing ERMS (Student’s t-test). Error bars denote +/- STD. Scale bars equal 100 μm (A). Not significant by Student’s t-test (n.s.).

https://doi.org/10.7554/eLife.19214.005
Figure 2 with 1 supplement
Tumors that transgenically express myf5 are fully transformed and retain a differentiated phenotype following engraftment into recipient animals.

(AF) Analysis of ERMS arising in transplanted fish. kRASG12D expressing ERMS arising in rag2E450fs transplant recipient fish (AC) compared with those that express both kRASG12D and mylpfa:myf5 (DF). Tumors were created in stable transgenic myf5:GFP/mylpfa:mCherry transgenic, AB-strain zebrafish and imaged following engraftment into recipient fish at 30 days post transplantation (dpt). Hematoxylin and eosin stained sections of representative tumors (B,E) and quantification of differentiation within individual ERMS (C,F; 1-less differentiated and 3-most differentiated). Asterisks denote p<0.001 by Chi-square test. (G,H) Representative flow cytometry analysis of fluorescently-labeled ERMS cells isolated from transplanted rag2E450fs zebrafish. (I) Graphical summary of ERMS cell sub-fractions that grow following engraftment into immune-deficient rag2E450fs recipients. Individual tumors are represented as separate bars with the proportion of G+ (green), G+R+ (yellow) and R+ (red) sub-populations denoted. **p=0.006. (J) Kaplan-Meijer analysis showing time-to-tumor onset in transplanted ERMS arising in rag2E450fs zebrafish (p=0.046, Log-rank Statistic, 2 × 105 cells/fish, N > 12 animals per arm, representing ≥3 independently-arising primary ERMS). (K) Relative tumor size at 30 days post engraftment (same animals analyzed as in J). (L) ERMS cells were also more differentiated following engraftment of myf5:GFP/mylpfa:mCherry ERMS cells into syngeneic recipient fish (p<0.001, Student’s T-test, N ≥ 3 independently arising primary ERMS and assessed in n ≥ 2 animals per transplanted tumor). Scale bars equal 2 mm (A,D) and 50 μm (B,E).

https://doi.org/10.7554/eLife.19214.006
Figure 2—figure supplement 1
Histological classification of transplanted zebrafish ERMS based on differentiation score.

Representative H and E-stained sections of zebrafish ERMS assigned to each differentiation category. Scale bars equal 100 μm.

https://doi.org/10.7554/eLife.19214.007
Figure 3 with 1 supplement
Limiting dilution cell transplantation shows that myf5 can confer tumor-propagating ability to differentiated myf5:GFP+/mylpfa:mCherry+ cells.

Tumors were generated in myf5:GFP/mylpfa:mCherry CG1-strain syngeneic zebrafish. Representative tumors arising in primary transplanted fish (1°T, AC) or secondary transplanted fish following engraftment with highly purified myf5:GFP+, mylpfa:mCherry-negative (2°T G+, DF) or myf5:GFP+, mylpfa:mCherry+ ERMS cells (2°T G+R+, GI). Sort purity following FACS is noted in the lower left panels of D and G and was >92% for each population. These cells were used for cell transplantations and data provided in D-I. Cell viability was >95%. (J,L) Graphical summary of tumor engraftment following limiting dilution cell transplantation using highly purified sorted ERMS cells. Data is combined from all tumors shown in Table 1. ***p<0.0002 by ELDA analysis. (K,M) Relative gene expression analysis of sorted G+ or G+R+ ERMS cells from representative kRASG12D (K) or kRASG12D; mylpfa:myf5 (M) expressing ERMS (Standard Deviation, n = 3 technical replicates per PCR condition). *p<0.05; **p<0.01 and ***p<0.001 by Student’s t-test.

https://doi.org/10.7554/eLife.19214.008
Figure 3—figure supplement 1
Analysis of transplanted ERMS arising in CG1-strain syngeneic recpients.

(A,D) Representative images of transplanted fish. ERMS were created in myf5-GFP/mylpfa-mCherry transgenic, CG1-strain syngeneic zebrafish and imaged following 30 days of engraftment. Genotypes denoted to the left. (B,E) Representative histology of transplanted tumors. (C,F) Quantification of differentiation based on histological review (1-less differentiated and 3-most differentiated). **p<0.01 by Chi-square test. (GP) Representative examples of sort purity following FACS for cells used in limiting dilution cell transplantation experiments. (GK) Sort purity following FACS for a representative kRASG12D-alone expressing ERMS and (LP) kRASG12D+ mylpfa:myf5 expressing ERMS (data is reproduced in lower left panels of Figure 3D and G). Scale bars equal 2 mm (A,D) and 100 μm (B,E).

https://doi.org/10.7554/eLife.19214.009
Figure 4 with 4 supplements
MYF5 and MYOD are required for human ERMS proliferation and growth.

(AB) Pearson correlation for gene expression of myogenic genes in primary human RMS as assessed by microarray (A) or RNA-sequencing (B). Heatmap represents correlation coefficients. (C) Western blot analysis for MYF5 and MYOD in human RMS cell lines. (DI) Rh18 ERMS cells following MYF5 knockdown with siRNA (DF) or shRNA (GI). (JO) RD ERMS cells following MYOD knockdown with siRNA (JL) or shRNA (MO). Western blot analysis following knockdown at 48 hr (D,J) and 72 hr (G,M). EdU and Propidium Iodide (PI) cell cycle analysis assessed by flow cytometry at 48 hr (E,F,K,L) and 72 hr (H,I,N,O). Standard Deviation denoted in FACS plots and graphs. Analysis shown in D-O was completed as technical replicates and completed ≥3 independent times with similar results. ***p<0.001 by Student’s t-test.

https://doi.org/10.7554/eLife.19214.011
Figure 4—figure supplement 1
Pearson correlation of gene expression from RNA-seq data of primary human RMS.

(A) Analysis of RNA-seq data from primary fusion-negative RMS (FN-RMS, N = 70) and (B) fusion-positive RMS (FP-RMS; N = 33 samples).

https://doi.org/10.7554/eLife.19214.012
Figure 4—figure supplement 2
Immunofluorescence for MYF5 and MYOD in Rh18 and RD ERMS cell lines.

(A) Confocal microscopy images of DAPI and antibody immunofluorescence-staining of Rh18 cells treated with control siRNA or si-MYF5 for 72 hr. (B) Confocal microscopy images of DAPI and antibody immunofluorescence staining of RD cells treated with control siRNA or si-MYOD. Anti-MYOD (green) and anti-MYF5 (red) and counterstained with DAPI (blue). Merged image shown to right. Scale bar equals 100 μm. Arrows denote representative examples of MYF5+/MYOD-negative RH18 cells in (A) and MYF5-negative/MYOD+ RD cells in (B).

https://doi.org/10.7554/eLife.19214.013
Figure 4—figure supplement 3
MYF5 and MYOD are required for human RMS proliferation and growth in vitro.

(AE) Rh18 ERMS cells following MYF5 knockdown and (FJ) RD ERMS cells following MYOD knockdown. EdU/PI Flow cytometry analysis performed at 72 hr post transfection with shRNAs (A,F). AnnV Flow cytometry quantification performed at 96 hr post siRNA transfection (B,G) or shRNA transfection (CD). Quantitation of nuclei counts performed on shRNA treated cells at 96 hr post infection (E,H). (I,J) Sphere colony formation assays performed in RD ERMS cells. Representative images of sh-SCR, sh-MYOD #1 and sh-MYOD #2 treated cells (I), images denote growth when seeding at 1 × 104 cells/well). Quantification of total spheres formed following seeding with different numbers of cells/well (J). Analysis was completed as technical replicates and completed ≥3 independent times with similar results. *p<0.05; **p<0.01; ***p<0.001 by Student’s t-test. Scale bar equals 50 um.

https://doi.org/10.7554/eLife.19214.014
Figure 4—figure supplement 4
MYF5 and MYOD are each specifically required for human RMS proliferation and growth in vitro.

(AL) siMYOD knockdown effects in human RMS cell lines. (AD) 381T ERMS; (EH) RMS559 ERMS; (IL) Rh3 ARMS/FP-RMS cells. (A,E,I) Western Blot analysis performed at 48 hr post siRNA transfection. (B,F,J) FACS plots for EdU/PI staining of cells at 48 hr after si-RNA transfection. (C,G,K) Quantification of EDU results. (D,H,L) AnnexinV Flow Cytometric analysis performed at 96 hr post transfection with si-SCR control, si-MYF5 or si-MYOD. (M–T) MRF knockdown is specific to each expressed transcription factor. Western blot analysis of Rh18 (M), RMS559 (N), Rh3 (O) and 381T (P) cells following 48 hr of siRNA treatment. Quantitation of Edu/PI flow cytometric analysis for Rh18 (Q), RMS559 (R), Rh3 (S) and RD (T) cells following 48 hr of siRNA treatment. Knockdown effects for RD cells are shown in Figure 6E. Error bars denote +/-STD from three technical replicates. Experiments were replicated three times on different days, showing similar results. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by Student’s t-test. Not significant (n.s.).

https://doi.org/10.7554/eLife.19214.015
Figure 5 with 1 supplement
MYF5 and MYOD are required for human ERMS xenograft growth.

Xenograft growth in Rh18 (AE), RD (FJ), and RMS559 (KO) following knockdown with scramble control shRNA (sh-SCR) or shRNAs specific to MYF5 or MYOD. (A,F,K) Western blot analysis of shRNA expressing cells harvested for transplantation at 72 hr after lenti-viral shRNA knockdown. Percent knockdown compared to shRNA control is shown. (B,G,L) Luciferase bioluminescent imaging of a representative animal at the time of implantation (left panel) or at later time points (right panel). Control shRNA cells were implanted into left flank and knockdown cells into the right (N = 6 mice per shRNA). Intensity represents total luminescence units measured per region of interest (L.U.) (C,H,M) Quantification tumor volume when assessed by luciferase imaging. Relative luminescence units (R.L.U). (D,I,N) Representative images of mice at the time of necropsy, with excised tumors shown in lower panels. (E,J,O) Quantification of tumor weight at the time of necropsy. Tumors that could not be identified at time of necropsy were assigned a value of zero for this analysis. Standard Error of the Mean are denoted in graphs. **p<0.01; ***p<0.001 by Mann-Whitney non-parametric test. Scale bar equals 1 cm in D,I, and N.

https://doi.org/10.7554/eLife.19214.016
Figure 5—figure supplement 1
MYF5 and MYOD are required for human ERMS growth and maintenance following xenograft transplantation into NOD/SCID/IL2g null mice.

(AC) Quantification of tumor growth when assessed by luciferase bioluminescence imaging over time. Rh18 (A), RD (B), and RMS559 (C). N = 6 animals per analysis. Error bars denote Standard Error of the Mean (SEM). *p<0.05; **p<0.01; ***p<0.001 by Student’s t-test. (DI) Hematoxylin Eosin stained sections of representative tumors isolated from mice engrafted with shRNA expressing Rh18 (DE), RD (FG), and RMS559 (HI). Scale bars equal 50 um.

https://doi.org/10.7554/eLife.19214.017
Figure 6 with 2 supplements
MYF5 and MYOD bind common promoter and enhancer regions and induce genes involved in muscle development and cell cycle.

(AB) ChIP-seq analysis showing genomic regions bound by both MYOD in RD cells and MYF5 in RH18 cells. H3K27 acetylation (H3K27ac). (C) Gene ontology enrichment of gene regions bound by both MYOD in RD cells and MYF5 in RH18 cells. GO Biological Processes, GO Cellular Component predictions, and binomial p-values denoted. (D) Signal tracks for ChIP-seq and RNA-seq surrounding MYOG (top) and CCND2 (bottom). Numbers to the right indicate reads per million mapped reads. (E) Quantitative real-time PCR gene expression analysis of RH18 (top) and RD cells (bottom). Cells were assessed following siRNA-mediated knockdown at 2 days (2dpt, blue bars) or 3 days post-transfection (three dpt, red bars). Error bars denote standard deviation. Student’s t-test; *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.19214.018
Figure 6—figure supplement 1
MYF5 and MYOD bind common promoter and enhancer regions.

(A) ChIP-seq identified genomic locations bound by MYOD in RD cells, MYF5 in RH18 cells, and H3K27 acetylation (H3K27ac). Common binding sites are denoted by boxed region at the top and reproduced in Figure 6. (B) Signal tracks for ChIP-seq and RNA-seq surrounding CDH15. Numbers to the right indicate reads per million mapped reads.

https://doi.org/10.7554/eLife.19214.019
Figure 6—figure supplement 2
Ccnd2a expression in zebrafish ERMS.

Quantitative real-time PCR gene expression performed on bulk zebrafish ERMS cells, comparing ccnd2a expression in zebrafish ERMS that express kRASG12D alone (K, N = 4) or co-express mylpfa:myf5 (K+M, N = 5). Average gene expression with 50% confidence intervals denoted by box. Mean, maximum, and minimum also denoted. Three independent primer pairs confirm a trend toward higher ccnd2a expression in mylpfa:myf5 expressing ERMS.

https://doi.org/10.7554/eLife.19214.020

Tables

Table 1

myf5 confers tumor-propagating ability to differentiated myf5-GFP+/mylpfa-mCherry+ ERMS cells. Engrafted animals per cell dose are noted. Experiments for three independent tumors are shown. G+ (myf5-GFP+/mylpfa-mCherry-), G+R+ (myf5-GFP+/mylpfa-mCherry+), R+ (myf5-GFP-/mylpfa-mCherry+), DN (myf5-GFP-/mylpfa-mCherry-). Not applicable (NA); tumor-propagating cell frequency (TPC Freq.); 95% confidence interval (95% CI). Lower panel denotes cumulative TPC frequency for all three ERMS analyzed per genotype. Asterisk denotes p=0.0002 by ELDA analysis.

https://doi.org/10.7554/eLife.19214.010

kRASG12D Tumor #1

kRASG12D + mylpfa:myf5 Tumor #1

Cell #

G+

G+R+

R+

DN

Cell #

G+

G+R+

R+

DN

1000

6 of 6

2 of 7

0 of 6

0 of 7

1000

2 of 3

4 of 5

0 of 6

0 of 6

100

5 of 9

0 of 9

0 of 8

0 of 10

100

6 of 10

2 of 10

0 of 8

0 of 7

10

0 of 8

0 of 8

0 of 9

0 of 7

10

3 of 10

1 of 10

0 of 10

0 of 8

TPC Freq.

1 in 140

1 in 3561

NA

NA

TPC Freq.

1 in 81

1 in 477

NA

NA

95% CI

59–329

872–13740

NA

NA

95% CI

40–165

201–1129

NA

NA

kRASG12D Tumor #2

kRASG12D + mylpfa:myf5 Tumor #2

Cell #

G+

G+R+

R+

DN

Cell #

G+

G+R+

R+

DN

1000

6 of 6

0 of 6

0 of 6

0 of 6

1000

2 of 3

1 of 2

0 of 7

0 of 7

100

4 of 7

2 of 10

0 of 10

0 of 10

100

1 of 6

3 of 6

0 of 7

0 of 10

10

1 of 8

0 of 9

0 of 10

0 of 8

10

0 of 8

0 of 9

0 of 10

0 of 8

TPC Freq.

1 in 109

1 in 3495

NA

NA

TPC Freq.

1 in 809

1 in 467

NA

NA

95% CI

44–270

808–15120

NA

NA

95% CI

244–2685

137–1589

NA

NA

kRASG12D Tumor #3

kRASG12D + mylpfa:myf5 Tumor #3

Cell #

G+

G+R+

R+

DN

Cell #

G+

G+R+

R+

DN

1000

2 of 3

0 of 2

0 of 3

0 of 4

1000

2 of 3

3 of 5

0 of 3

0 of 3

100

8 of 9

0 of 8

0 of 8

1 of 8

100

3 of 10

1 of 10

0 of 9

0 of 10

10

1 of 8

0 of 9

0 of 9

0 of 9

10

0 of 10

0 of 10

0 of 10

0 of 10

TPC Freq.

1 in 159

NA

NA

1 in 4840

TPC Freq.

1 in 530

1 in 1080

NA

NA

95% CI

63–401

NA

NA

632–37094

95% CI

194–1445

395–2957

NA

NA

Cumulative TPC frequency kRASG12D

Cumulative TPC frequency kRASG12D + mylpfa:myf5

Cell #

G+

G+R+

R+

DN

Cell #

G+

G+R+

R+

DN

TPC Freq.

1 in 146

1 in 4206

NA

NA

TPC Freq.

1 in 377

1 in 639*

NA

NA

95% CI

87–245

1550–11409

NA

NA

95% CI

212–670

363–1125

NA

NA

Additional files

Supplementary file 1

Primers and shRNAs used in this work.

https://doi.org/10.7554/eLife.19214.021
Supplementary file 2

GREAT analysis identifies commonly bound genomic sites between MYF5 and MYOD in human Rh18 and RD cells.

https://doi.org/10.7554/eLife.19214.022
Supplementary file 3

Common genomic regions bound by MYF5 and MYOD in ERMS that comprise the ‘cyclin-dependent protein kinase holoenzyme complex’ module.

https://doi.org/10.7554/eLife.19214.023
Supplementary file 4

Common genomic regions bound by MYF5 and MYOD in ERMS that comprise the genes found in the ‘embryonic skeletal system development’ module.

https://doi.org/10.7554/eLife.19214.024

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