Histone methyltransferase KMT1A restrains entry of alveolar rhabdomyosarcoma cells into a myogenic differentiated state

Abstract

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric muscle cancer, which arrested during the process of skeletal muscle differentiation. In muscle myoblast cells, ectopic expression of the histone H3 lysine 9 (H3K9) methytransferase KMT1A blocks differentiation by repressing a myogenic gene expression program. In this study, we tested the hypothesis that activation of a KMT1A-mediated program of transcriptional repression prevents ARMS cells from differentiating. We investigated whether KMT1A represses the expression of differentiation-associated genes in ARMS cells, thereby blocking muscle differentiation. Our results show that expression of KMT1A is induced in human ARMS cancer cell lines when cultured under differentiation-permissible conditions. shRNA-mediated knockdown of KMT1A decreased anchorage dependent and independent cell proliferation and tumor xenograft growth, increased expression of differentiation-associated genes, and promoted the appearance of a terminally differentiated-like phenotype. Finally, shRNA-directed KMT1A knockdown restored the impaired transcriptional activity of the myogenic regulator MyoD. Together, our results suggested that high levels of KMT1A in ARMS cells under differentiation conditions impairs MyoD function, thereby arresting myogenic differentiation in these tumor cells. Thus, targeting KMT1A may be a novel strategy for the treatment of this disease.

Figure 1

Expression of KMT1A and its HMTase activity are induced in ARMS cells under differentiation conditions (A) Semi-quantitative RT-PCR analysis of KMT1A and β-actin mRNA levels from total RNA extracted from indicated ARMS cells cultured as in (A). Relative KMT1A mRNA expression is represented as bar graphs following normalization with β-actin. Error bars, ± SEM (n=3). (B) Immunoblot of extracts from indicated ARMS cells cultured in GM or DM for 2 days, probed for KMT1A and β-actin. Relative KMT1A protein expression is represented as bar graph following normalization with β-actin. Error bars, ± SEM (n=3). (C) HMTase activity assay for NRIgG or KMT1A immunoprecipitates from extracts used in (A). After completion, reaction mixture was divided equally into two parts. One part was analyzed for detecting methylated and total input of GST-H3 (N) by fluorography and coomassie, respectively, and another part was subjected to immunoblotting, probed for KMT1A.

Figure 2

KMT1A shRNA in ARMS cells promotes growth inhibition and blocks tumor development. ARMS cells were transduced with lentivirus expressing indicated shRNA. Growth of shRNA-transduced and untransduced cells was evaluated for a period of 5 days cultured in GM (A) or 2 days in GM followed by 3 days in DM (B). Cell numbers at each time point are represented as ± SEM (n=3). (C) ARMS cells expressing indicated shRNA were grown in soft-agar. The number of soft-agar colonies were counted and presented ± SEM (n=3)). (D) Tumor growth was evaluated in nude mice injected with Rh28 cells transduced with lentivirus expressing scrambled shRNA (10 mice) or KMT1A shRNA (10 mice). Tumor growth was measured every 3 days for 21 days. Values are expressed as mean tumor volume, error bars, ± SEM (n=10) (E) Photographs of representative tumors that developed in 3 individual nude mice from the scrambled shRNA group (upper panel) or area surrounding the injection site for KMT1A shRNA-tranduced Rh28 cells (lower panel; - represents site of injection).

Figure 3

KMT1A down-regulation restores MyoD-dependent growth inhibition and transcriptional activation in ARMS cells. (A) Immunoblot of extracts from indicated shRNA-transduced Rh28 cells grown in GM, probed for KMT1A, MyoD and β-actin. (B) Growth of indicated shRNA-transduced Rh28 cells was evaluated for a period of 3 days grown in DM. Cell numbers at each time point are expressed as ± SEM (n=3). (C) Luciferase activity was evaluated in indicated shRNA-tranduced Rh30-4RE-luc cells cultured in DM for 2 days. Values were expressed after protein normalization. Error bars, ± SEM (n=3). (D) Immunoblot of extracts used in (C), probed for KMT1A, MyoD and β-actin.

Figure 4

Induction of myogenic differentiation in ARMS cells by KMT1A knock-down. (A) Immunoblot of extracts of ARMS cells expressing indicated shRNA cultured in DM for 2 days, probed for p21^cip1, myogenin, MyoD and GAPDH. (B) Immunoblot of extracts used in (A), probed for KMT1A and β-actin. (C) Immunoflurorescence image of MHC in ARMS cells expressing indicated shRNA cultured in DM for 7 and 10 days for Rh30 and Rh28 cells, respectively. The graph showed % MHC-positive cells and is represented as the mean from triplicate plates). (D) Cell morphology of indicated shRNA expressing ARMS cells visualized by phase-contrast microscopy.

Figure 5

Increased KMT1A HMTase activity associated with MyoD in ARMS cells under differentiation conditions. (A) NRIgG or KMT1A immunoprecipitates from extracts of indicated ARMS cells cultured in GM or DM for 2 days were immunoblotted with MyoD antibodies and re-probed for KMT1A. (B) Indicated immunoprecipitates from Rh30 cell extracts used in (A) were immunoblotted with KMT1A antibodies and re-probed for MyoD. (C) Indicated immunoprecipitates from extracts as in (A) were divided equally into two parts. One part was subjected to HMTase activity assay and processed as in Fig. 1C, and another part was immunoblotted with MyoD-specific antibody. (D) NRIgG or MyoD immunoprecipitates from extracts of ARMS cells expressing indicated shRNA cultured in DM for 2 days were subjected to HMT activity assay and processed as in Fig. 1C.

Figure 6

KMT1A and its activity-directed H3K9me3 mark are increased on the myogenin promoter in ARMS cells under differentiation conditions. (A) Using indicated antibodies, ChIP assays were performed on the myogenin promoter in ARMS cells cultured in GM or DM for 2 days. (B) Quantitative ChIP analysis using an anti-H3K9me3 antibody on the myogenin promoter in Rh30 cells grown as in (A), Error bar, ± SEM (n=3). (C) ChIP assays were performed as in (A), except chromatin from scramble shRNA expressing ARMS cells cultured in GM or DM for 2 days and KMT1A shRNA expressing these cells cultured in DM 2 days, and antibodies used were H3K9me3 and H3K9ac.