Ignatius MS et al. (2017), The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growt...

XB-ART-54815

Cell Rep 2017 Jun 13;1911:2304-2318. doi: 10.1016/j.celrep.2017.05.061.

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The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma.

Ignatius MS , Hayes MN , Lobbardi R , Chen EY , McCarthy KM , Sreenivas P , Motala Z , Durbin AD , Molodtsov A , Reeder S , Jin A , Sindiri S , Beleyea BC , Bhere D , Alexander MS , Shah K , Keller C , Linardic CM , Nielsen PG , Malkin D , Khan J , Langenau DM .

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Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.

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Species referenced: Xenopus
Genes referenced: ctrl hes1 hey1 kidins220 mef2c myf5 notch1 notch3 runx1 sms snai1 snai2 tbp

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	Figure 2. Notch1 Pathway Activation Confers Tumor-Propagating Activity to Differentiated ERMS Cells(A) Schematic of limiting dilution cell transplantation assay used to assess engraftment potential of fluorescently labeled ERMS cell fractions.(B–D) Engraftment with FACS-sorted myf5-GFP+/mylz2-mCherry-negative cells. (B) Whole animal image, (C) engrafted tumor cells analyzed by FACS, and (D) histology. Sort purity is denoted in the lower left corner of (B).(E–G) Engraftment with FACS-sorted double-positive myf5-GFP+/mylz2-mCherry+ differentiated cells. (E) Whole animal image, (F) engrafted tumor cells analyzed by FACS, and (G) histology. Sort purity denoted in lower left corner of (E).(H) Table showing combined analysis of engraftment rates for myf5-GFP+/mylz2-mCherry-negative, double-positive myf5-GFP+/mylz2-mCherry+, myf5-GFP-negative/mylz2-mCherry+, and double-negative cells. Number of tumors analyzed per condition is noted. ND, not determined; CI, confidence interval; inf, infinity.Scale bar in (B), also pertaining to (E), 2 mm; scale bar in (D), also pertaining to (G), 50 μm. See also Figure S2, Table S2, and Table S3.
	Figure 3. NOTCH1 Regulates Cell Growth, Differentiation, and Self-Renewal in Human ERMS(A) Microarray gene expression analysis of NOTCH1 in human skeletal muscle and rhabdomyosarcoma.(B) qPCR gene expression of NOTCH1 performed on skeletal muscle (SM), human rhabdomyosarcoma cell lines, and primary rhabdomyosarcoma.(C) Kaplan-Meijer analysis comparing survival in high versus low NOTCH1 expression in rhabdomyosarcoma patients (p = 0.013, log-rank statistic, combined analysis of ARMS and ERMS, n = 128).(D) Western blot analysis of RD cells following control scrambled shRNA (Scr) or NOTCH1 knockdown using three independent lentiviral shRNA hairpins (sh). Percent knockdown is noted.(E–H) Morphology of RD cells after 5 days of shRNA treatment. (E) Control (Scr) and (F–H) NOTCH1 knockdown.(I) Western blot analysis showing ICN1 expression in stable RD knockdown cells. Percent knockdown is noted.(J) qPCR gene expression for a panel of muscle differentiation genes in control (Scr) and NOTCH1 knockdown RD cells.(K and L) Immunofluorescence staining for myosin heavy chain in stable RD cells expressing (K) scrambled or (L) NOTCH1 shRNA #1. Percentages of tumor cells with myosin heavy chain expression are denoted ± SD.(M) Quantitation of the percentage of tumor cells with myosin heavy chain expression in RD cells treated with control and two NOTCH1 shRNAs.(N–Q) Sphere formation in stable RD cells. Images of spheres from (N) scrambled or (O) NOTCH1 shRNA#1 knockdown cells. Quantitation of sphere colony formation when assessed at varying cell numbers for (P) NOTCH1 shRNA#1 or (Q) shRNA #2.(R) Sphere formation in human PDX PCB00234 ERMS cells treated with DMSO or Notch pathway inhibitor DBZ and assessed at 15 days.(S) Top: western blot analysis of RD cells with and without NOTCH1ΔE. Bottom: RD cells treated with DBZ for 10 days.(T–V) Sphere colony formation in RD cells treated with (T) DMSO and compared with RD cells expressing NOTCH1ΔE treated with (U) DMSO or (V) 5 μm DBZ.(W) Quantification of results.Scale bar in (E), also pertaining to (F)–(H), 200 μm; scale bar in (K), also pertaining to (L), 100 μm; scale bar in (N), also pertaining to (O), (T), and (V), 400 μm. Asterisks denote statistical differences by Student's t test (p < 0.05; p < 0.01; **p < 0.001). Error bars represent ±1 SD. See also Figure S3.
	Figure 4. SNAIL1 Is a Downstream Target of NOTCH1 in Human ERMS(A) qPCR gene expression of stable shRNA control (Scr) and NOTCH1 knockdown SMS-CTR cells.(B) Western blot analysis of RD cells following stable knockdown of NOTCH1. Percent knockdown is noted. ICN1 blot is the same as in Figure 3I.(C) Western blot analysis showing ICN1 and SNAI1 co-expression across human rhabdomyosarcoma cell lines.(D) Pearson correlation between the expression of NOTCH1, NOTCH3, SNAI1, SNAI2, HES1, HEY1, and MEF2C in primary human ERMS assessed by RNA-sequencing analysis.(E) qPCR gene expression of SNAI1 performed on skeletal muscle (SM), human rhabdomyosarcoma cell lines, and primary rhabdomyosarcoma. TBP, TATA box-binding protein.(F) ChIP assay in RD ERMS cells followed by qPCR gene expression for NOTCH1-binding regions in a region 1 kb upstream of the SNAI1 transcription start site (TSS). Ctrl, control; Chr, chromosome; NG, non geneic.Error bars in (A), (E), and (F) represent ±1 SD. In (A), p < 0.05. In (E), p < 0.01. In (F), p < 0.05; *p < 0.01, Student's t test; ns, not significant. See also Figure S4.
	Figure 5. SNAI1 Regulates Cell Growth, Differentiation, and Self-Renewal in Human ERMS(A) Western blot analysis of RD cells following control shRNA (Scr) or SNAI1 knockdown using three independent lentiviral shRNA hairpins. Percent knockdown is noted.(B–E) Morphology of RD cells after 5 days post of shRNA treatment. (B) Control (Scr) and (C–E) SNAI1 knockdown.(F) Western blot analysis showing SNAI1 expression in stable RD knockdown cells.(G) qPCR gene expression for a panel of muscle differentiation genes in RD knockdown cells.(H and I) Immunofluorescence staining for myosin heavy chain in stable RD cells expressing (H) control or (I) SNAI1 shRNA. Percentage of tumor cells with myosin heavy chain expression are denoted ± SD.(J) Quantitation of the percentage of tumor cells with myosin heavy chain expression in RD cells treated with control and SNAI1 shRNAs.(K–N) Sphere formation in stable RD cells. Images of spheres from (K) scrambled or (L) SNAI1 shRNA#1 knockdown cells. Quantitation of sphere colony formation when assessed at varying cell numbers for control shRNA, (M) SNAI1 shRNA #1, or (N) shRNA#2.(O) Sphere formation in RD cells stably expressing SNAI1-ERSS with and without 4-hydroxytamoxifen (4 OHT) treatment.(P) Western blot analysis of RD cells that stably express SNAI1-ERSS. Cells were treated for 10 days with DBZ and/or tamoxifen as noted.(Q–S) Sphere formation in RD cells expressing SNAI1-ERSS and treated for 10 days with (Q) DMSO, (R) DBZ, or (S) DBZ and tamoxifen as noted.(T) Quantification of data shown in (Q)–(S).Scale bar in (B), also pertaining to (C)–(E), 200 μm; scale bar in (H), also pertaining to (I), 100 μm; scale bar in (K), also pertaining to (L), (Q), (R), and (S), 400 μm. Asterisks denote significant differences based on Student's t test (p < 0.05; p < 0.01; **p < 0.001). Error bars indicate ±1 SD. See also Figures S4 and S5.
	Figure 6. NOTCH1 and SNAI1 Are Required for Growth and Maintenance of Human ERMS following Xenograft Transplantation into Mice(A–H) NOTCH1 knockdown suppresses RD growth in xenograft-transplanted mice. (A) Luciferase bioluminescent imaging of subcutaneously engrafted RD cells following stable shRNA knockdown and injection into the flanks of NOD/SCID/IL2g null mice. Scrambled (Scr; left) or NOTCH1 knockdown (right). Representative animal shown. d, days. (B) Quantitation of tumor growth. Error bars represent ±1 SD. (C) Quantitation of tumor weight following excision at necropsy (p < 0.0001, Fisher's exact test). Error bars are ± 1 SD. Representative tumors are shown at right. (D and E) Representative images of histology from engrafted tumors. (F–H) KI67 staining in (F) and (G) and quantification in (H).(I–P) SNAI1 knockdown suppresses RD growth in xenograft-transplanted mice. (I) Luciferase bioluminescent imaging of engrafted mice. Scrambled (Scr; left) or SNAI1 knockdown (right). (J) Quantitation of tumor growth. Error bars represent ± 1 SD. (K) Quantitation of tumor weight following excision at necropsy performed between 88 and 93 days post-transplantation (p < 0.0001, Fisher's exact test). Representative tumors are shown at right. Error bars are ± 1 SD. (L and M) Representative images of histology from engrafted tumors. (N–P) KI67 staining, in (N) and (O), and quantification, in (P), of the data shown in (N) and (O). Scale bar in (D), also pertaining to (E)–(G) and (L)–(O), 50 μm. Asterisks denote significant differences based on Student's t test (p < 0.01; *p < 0.001). NS, not significant.See also Figure S6.
	Figure 7. The NOTCH1/SNAI1 Signaling Axis Suppresses MEF2C to Block Differentiation and to Elevate Human ERMS Self-Renewal(A) qPCR for MEF2C, RUNX1, and JDP2 following NOTCH1 knockdown in RD cells. Scr, scrambled.(B) qPCR expression following SNAI1 knockdown in RD cells.(C) Microarray gene expression analysis of MEF2C in human skeletal muscle (SM) and rhabdomyosarcoma.(D) Immunofluorescence staining of RD- and RD+ NOTCH1ΔE-expressing cells. Red indicates MEF2C, green indicates myosin heavy chain, and blue indicates DAPI.(E) Quantification of the percentage of MEF2C-positive RD cells following stable knockdown with scrambled or NOTCH1 shRNA.(F) Quantification of the percentage of MEF2C-positive RD cells following knockdown with SNAI1 shRNA.(G) Immunofluorescence staining performed on NOTCH1 knockdown cells following treatment with control siRNA or MEF2C siRNA.(H) Western blot analysis of stable NOTCH1 knockdown cells following treatment with control siRNA or MEF2C siRNA.(I) Immunofluorescence staining following doxycycline-inducible MEF2C expression. -DOX, no doxycycline; +DOX, with doxycycline. OE, over expression.(J) Western blot analysis of human RD ERMS cells that have doxycycline-inducible expression of MEF2C.(K) Sphere formation in ERMS RD cells following doxycycline-inducible expression of MEF2C. Spheres assessed at 10 days of culture with colony number per 10,000 seeded cells are noted (±SD; p < 0.01).(L) Western blot analysis of RD cells that stably express SNAI1-ERSS cells. Cells were treated with 1 μM 4-hydroxytamoxifen to turn on SNAI1 activity and then were treated with DMSO or DBZ for 10 days in culture.Scale bar in (D), also pertaining to (G) and (I), 50 μm; scale bar in (K), 400 μm. Asterisks denote significant differences based on Student's t test: p < 0.05; p < 0.01; **p < 0.001.See also Figure S7.

References [+] :

Beck, Unravelling cancer stem cell potential. 2013, Pubmed

	Figure 2. Notch1 Pathway Activation Confers Tumor-Propagating Activity to Differentiated ERMS Cells(A) Schematic of limiting dilution cell transplantation assay used to assess engraftment potential of fluorescently labeled ERMS cell fractions.(B–D) Engraftment with FACS-sorted myf5-GFP+/mylz2-mCherry-negative cells. (B) Whole animal image, (C) engrafted tumor cells analyzed by FACS, and (D) histology. Sort purity is denoted in the lower left corner of (B).(E–G) Engraftment with FACS-sorted double-positive myf5-GFP+/mylz2-mCherry+ differentiated cells. (E) Whole animal image, (F) engrafted tumor cells analyzed by FACS, and (G) histology. Sort purity denoted in lower left corner of (E).(H) Table showing combined analysis of engraftment rates for myf5-GFP+/mylz2-mCherry-negative, double-positive myf5-GFP+/mylz2-mCherry+, myf5-GFP-negative/mylz2-mCherry+, and double-negative cells. Number of tumors analyzed per condition is noted. ND, not determined; CI, confidence interval; inf, infinity.Scale bar in (B), also pertaining to (E), 2 mm; scale bar in (D), also pertaining to (G), 50 μm. See also Figure S2, Table S2, and Table S3.
	Figure 3. NOTCH1 Regulates Cell Growth, Differentiation, and Self-Renewal in Human ERMS(A) Microarray gene expression analysis of NOTCH1 in human skeletal muscle and rhabdomyosarcoma.(B) qPCR gene expression of NOTCH1 performed on skeletal muscle (SM), human rhabdomyosarcoma cell lines, and primary rhabdomyosarcoma.(C) Kaplan-Meijer analysis comparing survival in high versus low NOTCH1 expression in rhabdomyosarcoma patients (p = 0.013, log-rank statistic, combined analysis of ARMS and ERMS, n = 128).(D) Western blot analysis of RD cells following control scrambled shRNA (Scr) or NOTCH1 knockdown using three independent lentiviral shRNA hairpins (sh). Percent knockdown is noted.(E–H) Morphology of RD cells after 5 days of shRNA treatment. (E) Control (Scr) and (F–H) NOTCH1 knockdown.(I) Western blot analysis showing ICN1 expression in stable RD knockdown cells. Percent knockdown is noted.(J) qPCR gene expression for a panel of muscle differentiation genes in control (Scr) and NOTCH1 knockdown RD cells.(K and L) Immunofluorescence staining for myosin heavy chain in stable RD cells expressing (K) scrambled or (L) NOTCH1 shRNA #1. Percentages of tumor cells with myosin heavy chain expression are denoted ± SD.(M) Quantitation of the percentage of tumor cells with myosin heavy chain expression in RD cells treated with control and two NOTCH1 shRNAs.(N–Q) Sphere formation in stable RD cells. Images of spheres from (N) scrambled or (O) NOTCH1 shRNA#1 knockdown cells. Quantitation of sphere colony formation when assessed at varying cell numbers for (P) NOTCH1 shRNA#1 or (Q) shRNA #2.(R) Sphere formation in human PDX PCB00234 ERMS cells treated with DMSO or Notch pathway inhibitor DBZ and assessed at 15 days.(S) Top: western blot analysis of RD cells with and without NOTCH1ΔE. Bottom: RD cells treated with DBZ for 10 days.(T–V) Sphere colony formation in RD cells treated with (T) DMSO and compared with RD cells expressing NOTCH1ΔE treated with (U) DMSO or (V) 5 μm DBZ.(W) Quantification of results.Scale bar in (E), also pertaining to (F)–(H), 200 μm; scale bar in (K), also pertaining to (L), 100 μm; scale bar in (N), also pertaining to (O), (T), and (V), 400 μm. Asterisks denote statistical differences by Student's t test (p < 0.05; p < 0.01; **p < 0.001). Error bars represent ±1 SD. See also Figure S3.
	Figure 4. SNAIL1 Is a Downstream Target of NOTCH1 in Human ERMS(A) qPCR gene expression of stable shRNA control (Scr) and NOTCH1 knockdown SMS-CTR cells.(B) Western blot analysis of RD cells following stable knockdown of NOTCH1. Percent knockdown is noted. ICN1 blot is the same as in Figure 3I.(C) Western blot analysis showing ICN1 and SNAI1 co-expression across human rhabdomyosarcoma cell lines.(D) Pearson correlation between the expression of NOTCH1, NOTCH3, SNAI1, SNAI2, HES1, HEY1, and MEF2C in primary human ERMS assessed by RNA-sequencing analysis.(E) qPCR gene expression of SNAI1 performed on skeletal muscle (SM), human rhabdomyosarcoma cell lines, and primary rhabdomyosarcoma. TBP, TATA box-binding protein.(F) ChIP assay in RD ERMS cells followed by qPCR gene expression for NOTCH1-binding regions in a region 1 kb upstream of the SNAI1 transcription start site (TSS). Ctrl, control; Chr, chromosome; NG, non geneic.Error bars in (A), (E), and (F) represent ±1 SD. In (A), p < 0.05. In (E), p < 0.01. In (F), p < 0.05; *p < 0.01, Student's t test; ns, not significant. See also Figure S4.
	Figure 5. SNAI1 Regulates Cell Growth, Differentiation, and Self-Renewal in Human ERMS(A) Western blot analysis of RD cells following control shRNA (Scr) or SNAI1 knockdown using three independent lentiviral shRNA hairpins. Percent knockdown is noted.(B–E) Morphology of RD cells after 5 days post of shRNA treatment. (B) Control (Scr) and (C–E) SNAI1 knockdown.(F) Western blot analysis showing SNAI1 expression in stable RD knockdown cells.(G) qPCR gene expression for a panel of muscle differentiation genes in RD knockdown cells.(H and I) Immunofluorescence staining for myosin heavy chain in stable RD cells expressing (H) control or (I) SNAI1 shRNA. Percentage of tumor cells with myosin heavy chain expression are denoted ± SD.(J) Quantitation of the percentage of tumor cells with myosin heavy chain expression in RD cells treated with control and SNAI1 shRNAs.(K–N) Sphere formation in stable RD cells. Images of spheres from (K) scrambled or (L) SNAI1 shRNA#1 knockdown cells. Quantitation of sphere colony formation when assessed at varying cell numbers for control shRNA, (M) SNAI1 shRNA #1, or (N) shRNA#2.(O) Sphere formation in RD cells stably expressing SNAI1-ERSS with and without 4-hydroxytamoxifen (4 OHT) treatment.(P) Western blot analysis of RD cells that stably express SNAI1-ERSS. Cells were treated for 10 days with DBZ and/or tamoxifen as noted.(Q–S) Sphere formation in RD cells expressing SNAI1-ERSS and treated for 10 days with (Q) DMSO, (R) DBZ, or (S) DBZ and tamoxifen as noted.(T) Quantification of data shown in (Q)–(S).Scale bar in (B), also pertaining to (C)–(E), 200 μm; scale bar in (H), also pertaining to (I), 100 μm; scale bar in (K), also pertaining to (L), (Q), (R), and (S), 400 μm. Asterisks denote significant differences based on Student's t test (p < 0.05; p < 0.01; **p < 0.001). Error bars indicate ±1 SD. See also Figures S4 and S5.
	Figure 6. NOTCH1 and SNAI1 Are Required for Growth and Maintenance of Human ERMS following Xenograft Transplantation into Mice(A–H) NOTCH1 knockdown suppresses RD growth in xenograft-transplanted mice. (A) Luciferase bioluminescent imaging of subcutaneously engrafted RD cells following stable shRNA knockdown and injection into the flanks of NOD/SCID/IL2g null mice. Scrambled (Scr; left) or NOTCH1 knockdown (right). Representative animal shown. d, days. (B) Quantitation of tumor growth. Error bars represent ±1 SD. (C) Quantitation of tumor weight following excision at necropsy (p < 0.0001, Fisher's exact test). Error bars are ± 1 SD. Representative tumors are shown at right. (D and E) Representative images of histology from engrafted tumors. (F–H) KI67 staining in (F) and (G) and quantification in (H).(I–P) SNAI1 knockdown suppresses RD growth in xenograft-transplanted mice. (I) Luciferase bioluminescent imaging of engrafted mice. Scrambled (Scr; left) or SNAI1 knockdown (right). (J) Quantitation of tumor growth. Error bars represent ± 1 SD. (K) Quantitation of tumor weight following excision at necropsy performed between 88 and 93 days post-transplantation (p < 0.0001, Fisher's exact test). Representative tumors are shown at right. Error bars are ± 1 SD. (L and M) Representative images of histology from engrafted tumors. (N–P) KI67 staining, in (N) and (O), and quantification, in (P), of the data shown in (N) and (O). Scale bar in (D), also pertaining to (E)–(G) and (L)–(O), 50 μm. Asterisks denote significant differences based on Student's t test (p < 0.01; *p < 0.001). NS, not significant.See also Figure S6.
	Figure 7. The NOTCH1/SNAI1 Signaling Axis Suppresses MEF2C to Block Differentiation and to Elevate Human ERMS Self-Renewal(A) qPCR for MEF2C, RUNX1, and JDP2 following NOTCH1 knockdown in RD cells. Scr, scrambled.(B) qPCR expression following SNAI1 knockdown in RD cells.(C) Microarray gene expression analysis of MEF2C in human skeletal muscle (SM) and rhabdomyosarcoma.(D) Immunofluorescence staining of RD- and RD+ NOTCH1ΔE-expressing cells. Red indicates MEF2C, green indicates myosin heavy chain, and blue indicates DAPI.(E) Quantification of the percentage of MEF2C-positive RD cells following stable knockdown with scrambled or NOTCH1 shRNA.(F) Quantification of the percentage of MEF2C-positive RD cells following knockdown with SNAI1 shRNA.(G) Immunofluorescence staining performed on NOTCH1 knockdown cells following treatment with control siRNA or MEF2C siRNA.(H) Western blot analysis of stable NOTCH1 knockdown cells following treatment with control siRNA or MEF2C siRNA.(I) Immunofluorescence staining following doxycycline-inducible MEF2C expression. -DOX, no doxycycline; +DOX, with doxycycline. OE, over expression.(J) Western blot analysis of human RD ERMS cells that have doxycycline-inducible expression of MEF2C.(K) Sphere formation in ERMS RD cells following doxycycline-inducible expression of MEF2C. Spheres assessed at 10 days of culture with colony number per 10,000 seeded cells are noted (±SD; p < 0.01).(L) Western blot analysis of RD cells that stably express SNAI1-ERSS cells. Cells were treated with 1 μM 4-hydroxytamoxifen to turn on SNAI1 activity and then were treated with DMSO or DBZ for 10 days in culture.Scale bar in (D), also pertaining to (G) and (I), 50 μm; scale bar in (K), 400 μm. Asterisks denote significant differences based on Student's t test: p < 0.05; p < 0.01; **p < 0.001.See also Figure S7.