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LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Ji X, Lu H, Zhou Q, Luo K - Elife (2014)

Bottom Line: The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors.A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex.Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

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P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.(A) An in vitro kinase assay using GST-CTD of Pol II as an exogenous substrate shows that silencing LARP7 results in activation of P-TEFb kinase. (B) Flavopiridol (Flvp) inhibits Ser2 phosphorylation of Pol II CTD in MCF10A and T47D cells. Cells were treated with varying concentrations of flavopiridol for 7 hr before lysis, and pSer2 and total Pol II levels were analyzed by Western blotting. Tubulin was used as a loading control. (C–D) Treatment of cells with 0.3 μM flavopiridol reverses the accelerated cell motility of shLARP7 cells in the wound healing assay (C) and cell migration in the Transwell assay (D). Data are presented as the mean ± SD. p values were determined by the Student's t test. *p<0.05; comparison was between shLARP7 and scramble groups under DMSO treatment; #p<0.05; comparison was between Flvp- and DMSO-treated cells within the same cell lines. (E) Silencing CDK9 by siRNA reverses shLARP7-induced increase in cell migration. Two siCDK9s (siCDK9-1 and siCDK9-2) were transfected into T47D shLARP7-2 cells, and cell migration was assessed by a Transwell assay and quantified in the graph below. The efficiency of CDK9 knockdown was examined by Western blotting (upper panel). p values were determined by the Student's t test. *p<0.05, compared between shLARP7-2 siCtrl and scramble; #p<0.05, compared between shCDK9s and siCtrl.DOI:http://dx.doi.org/10.7554/eLife.02907.009
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fig4: P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.(A) An in vitro kinase assay using GST-CTD of Pol II as an exogenous substrate shows that silencing LARP7 results in activation of P-TEFb kinase. (B) Flavopiridol (Flvp) inhibits Ser2 phosphorylation of Pol II CTD in MCF10A and T47D cells. Cells were treated with varying concentrations of flavopiridol for 7 hr before lysis, and pSer2 and total Pol II levels were analyzed by Western blotting. Tubulin was used as a loading control. (C–D) Treatment of cells with 0.3 μM flavopiridol reverses the accelerated cell motility of shLARP7 cells in the wound healing assay (C) and cell migration in the Transwell assay (D). Data are presented as the mean ± SD. p values were determined by the Student's t test. *p<0.05; comparison was between shLARP7 and scramble groups under DMSO treatment; #p<0.05; comparison was between Flvp- and DMSO-treated cells within the same cell lines. (E) Silencing CDK9 by siRNA reverses shLARP7-induced increase in cell migration. Two siCDK9s (siCDK9-1 and siCDK9-2) were transfected into T47D shLARP7-2 cells, and cell migration was assessed by a Transwell assay and quantified in the graph below. The efficiency of CDK9 knockdown was examined by Western blotting (upper panel). p values were determined by the Student's t test. *p<0.05, compared between shLARP7-2 siCtrl and scramble; #p<0.05, compared between shCDK9s and siCtrl.DOI:http://dx.doi.org/10.7554/eLife.02907.009

Mentions: Because LARP7 is a critical component of the inhibitory 7SK snRNP, we hypothesize that its ability to suppress tumor progression is due to the sequestration of P-TEFb in 7SK snRNP. To test this hypothesis, we first examined the kinase activity of P-TEFb upon LARP7 KD in an in vitro kinase assay using recombinant GST-CTD as a substrate. As predicted, the ability of endogenous CDK9 to phosphorylate the CTD was dramatically higher in the KD cells than in the control cells (Figure 4A).10.7554/eLife.02907.009Figure 4.P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.


LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Ji X, Lu H, Zhou Q, Luo K - Elife (2014)

P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.(A) An in vitro kinase assay using GST-CTD of Pol II as an exogenous substrate shows that silencing LARP7 results in activation of P-TEFb kinase. (B) Flavopiridol (Flvp) inhibits Ser2 phosphorylation of Pol II CTD in MCF10A and T47D cells. Cells were treated with varying concentrations of flavopiridol for 7 hr before lysis, and pSer2 and total Pol II levels were analyzed by Western blotting. Tubulin was used as a loading control. (C–D) Treatment of cells with 0.3 μM flavopiridol reverses the accelerated cell motility of shLARP7 cells in the wound healing assay (C) and cell migration in the Transwell assay (D). Data are presented as the mean ± SD. p values were determined by the Student's t test. *p<0.05; comparison was between shLARP7 and scramble groups under DMSO treatment; #p<0.05; comparison was between Flvp- and DMSO-treated cells within the same cell lines. (E) Silencing CDK9 by siRNA reverses shLARP7-induced increase in cell migration. Two siCDK9s (siCDK9-1 and siCDK9-2) were transfected into T47D shLARP7-2 cells, and cell migration was assessed by a Transwell assay and quantified in the graph below. The efficiency of CDK9 knockdown was examined by Western blotting (upper panel). p values were determined by the Student's t test. *p<0.05, compared between shLARP7-2 siCtrl and scramble; #p<0.05, compared between shCDK9s and siCtrl.DOI:http://dx.doi.org/10.7554/eLife.02907.009
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fig4: P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.(A) An in vitro kinase assay using GST-CTD of Pol II as an exogenous substrate shows that silencing LARP7 results in activation of P-TEFb kinase. (B) Flavopiridol (Flvp) inhibits Ser2 phosphorylation of Pol II CTD in MCF10A and T47D cells. Cells were treated with varying concentrations of flavopiridol for 7 hr before lysis, and pSer2 and total Pol II levels were analyzed by Western blotting. Tubulin was used as a loading control. (C–D) Treatment of cells with 0.3 μM flavopiridol reverses the accelerated cell motility of shLARP7 cells in the wound healing assay (C) and cell migration in the Transwell assay (D). Data are presented as the mean ± SD. p values were determined by the Student's t test. *p<0.05; comparison was between shLARP7 and scramble groups under DMSO treatment; #p<0.05; comparison was between Flvp- and DMSO-treated cells within the same cell lines. (E) Silencing CDK9 by siRNA reverses shLARP7-induced increase in cell migration. Two siCDK9s (siCDK9-1 and siCDK9-2) were transfected into T47D shLARP7-2 cells, and cell migration was assessed by a Transwell assay and quantified in the graph below. The efficiency of CDK9 knockdown was examined by Western blotting (upper panel). p values were determined by the Student's t test. *p<0.05, compared between shLARP7-2 siCtrl and scramble; #p<0.05, compared between shCDK9s and siCtrl.DOI:http://dx.doi.org/10.7554/eLife.02907.009
Mentions: Because LARP7 is a critical component of the inhibitory 7SK snRNP, we hypothesize that its ability to suppress tumor progression is due to the sequestration of P-TEFb in 7SK snRNP. To test this hypothesis, we first examined the kinase activity of P-TEFb upon LARP7 KD in an in vitro kinase assay using recombinant GST-CTD as a substrate. As predicted, the ability of endogenous CDK9 to phosphorylate the CTD was dramatically higher in the KD cells than in the control cells (Figure 4A).10.7554/eLife.02907.009Figure 4.P-TEFb is required for the increased cell migration and invasion induced by LARP7 KD.

Bottom Line: The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors.A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex.Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

Show MeSH
Related in: MedlinePlus