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Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors.

Algar EM, Muscat A, Dagar V, Rickert C, Chow CW, Biegel JA, Ekert PG, Saffery R, Craig J, Johnstone RW, Ashley DM - PLoS ONE (2009)

Bottom Line: We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter.We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression.Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Centre research laboratory, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia. elizabeth.algar@rch.org.au

ABSTRACT
SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

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SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines.(A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.
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pone-0004482-g001: SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines.(A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.

Mentions: G401 clones expressing SMARCB1 under the control of a tamoxifen inducible promoter system (pcDNA3 UAS SMARCB1 and pEF puro/GEV) were selected in geneticin and puromycin. Two G401 clones, F13 and F22, showing inducible SMARCB1 expression, were selected for further investigation. A pool of STM91-01 cells (STMpc), transduced with a lentiviral vector system (pF 5×UAS Sv40 puro/ini1 and pF GEV16) was also examined. SMARCB1 protein was readily detectable 24 hours after exposure to 1 uM 4HT in all cultures, (Figure 1A). Cell cycle analysis was performed on cultures of F22 cells 72 hours after induction of SMARCB1 protein expression. In SMARCB1 expressing cells 41.4+/−5% of cells were arrested in G0 compared with 7.0+/−1.4% in un-induced cells (Supplementary Figure S1A), consistent with observations made previously that SMARCB1 can induce cell cycle arrest [14]–[16]. In contrast, STMpc cells induced to express SMARCB1 showed little change in the cell cycle (Supplementary Figure S1A). One explanation for this is that the parental line, STM91-01, is derived from a RT lung metastasis and may have acquired additional oncogenic mutations and hence greater resistance to growth inhibitory signals. Reincke et al [21] also found this cell line difficult to transduce with recombinant adenovirus containing SMARCB1 and reportedly found inconsistent cell cycle changes following infection. CDKN1C mRNA was elevated in all lines (F22, F13, STMpc) following induction of SMARCB1 expression with 1 uM 4HT whereas other CDK family members including CDKN1A (p21/WAF1) and CDKN1B (p27) did not show uniform responses following 4HT treatment (Figure 1B). Quantitative analysis of CDKN1C expression by real-time PCR in F22 cells showed four-fold increases in CDKN1C expression 24 hours following SMARCB1 induction, and STMpc showed six-fold increases in CDKN1C expression (Figure 1C). 1 uM 4HT alone had a negligible effect on CDKN1C expression in parental G401 and STM91-01 cultures demonstrating that the observed effect on CDKN1C expression was directly attributable to the induction of SMARCB1 protein (results not shown). Endogenous levels of CDKN1C protein were low in un-induced F22 cells however following SMARCB1 induction, were increased to levels consistent with the proportional increase in CDKN1C transcript levels (Figure 1D).


Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors.

Algar EM, Muscat A, Dagar V, Rickert C, Chow CW, Biegel JA, Ekert PG, Saffery R, Craig J, Johnstone RW, Ashley DM - PLoS ONE (2009)

SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines.(A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637419&req=5

pone-0004482-g001: SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines.(A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.
Mentions: G401 clones expressing SMARCB1 under the control of a tamoxifen inducible promoter system (pcDNA3 UAS SMARCB1 and pEF puro/GEV) were selected in geneticin and puromycin. Two G401 clones, F13 and F22, showing inducible SMARCB1 expression, were selected for further investigation. A pool of STM91-01 cells (STMpc), transduced with a lentiviral vector system (pF 5×UAS Sv40 puro/ini1 and pF GEV16) was also examined. SMARCB1 protein was readily detectable 24 hours after exposure to 1 uM 4HT in all cultures, (Figure 1A). Cell cycle analysis was performed on cultures of F22 cells 72 hours after induction of SMARCB1 protein expression. In SMARCB1 expressing cells 41.4+/−5% of cells were arrested in G0 compared with 7.0+/−1.4% in un-induced cells (Supplementary Figure S1A), consistent with observations made previously that SMARCB1 can induce cell cycle arrest [14]–[16]. In contrast, STMpc cells induced to express SMARCB1 showed little change in the cell cycle (Supplementary Figure S1A). One explanation for this is that the parental line, STM91-01, is derived from a RT lung metastasis and may have acquired additional oncogenic mutations and hence greater resistance to growth inhibitory signals. Reincke et al [21] also found this cell line difficult to transduce with recombinant adenovirus containing SMARCB1 and reportedly found inconsistent cell cycle changes following infection. CDKN1C mRNA was elevated in all lines (F22, F13, STMpc) following induction of SMARCB1 expression with 1 uM 4HT whereas other CDK family members including CDKN1A (p21/WAF1) and CDKN1B (p27) did not show uniform responses following 4HT treatment (Figure 1B). Quantitative analysis of CDKN1C expression by real-time PCR in F22 cells showed four-fold increases in CDKN1C expression 24 hours following SMARCB1 induction, and STMpc showed six-fold increases in CDKN1C expression (Figure 1C). 1 uM 4HT alone had a negligible effect on CDKN1C expression in parental G401 and STM91-01 cultures demonstrating that the observed effect on CDKN1C expression was directly attributable to the induction of SMARCB1 protein (results not shown). Endogenous levels of CDKN1C protein were low in un-induced F22 cells however following SMARCB1 induction, were increased to levels consistent with the proportional increase in CDKN1C transcript levels (Figure 1D).

Bottom Line: We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter.We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression.Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Centre research laboratory, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia. elizabeth.algar@rch.org.au

ABSTRACT
SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

Show MeSH
Related in: MedlinePlus