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Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Roy SS, Gonugunta VK, Bandyopadhyay A, Rao MK, Goodall GJ, Sun LZ, Tekmal RR, Vadlamudi RK - Oncogene (2013)

Bottom Line: Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2.Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis.Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, UT Health Science Center, San Antonio, TX, USA.

ABSTRACT
Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

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MiRs influence the PELP1-mediated migratory and invasion potential. A, Boyden chamber analysis of the cell migration potential of the ZR cells transfected with mimetics and ZR-PELP1-shRNA cells transfected with inhibitors/antagomirs of miRs 200a and 141. B, MDA-MB-231 and MDA-MB-231-PELP1-shRNA cells were transfected with miRs 200a and 141 mimetics and inhibitors/antagomirs respectively and their effects on invasion were analyzed by using Matrigel invasion chamber assays. Mean and SDs are from three independent experiments. C, Western blot analysis of EMT genes expression in control, PELP1-shRNA cells and PELP1-shRNA clones treated with miR inhibitors/antagomirs. D, ZR-control-shRNA and ZR-PELP1-shRNA cells were transfected with ZEB1 and ZEB2 3′UTR reporter genes and treated with miR-200a and 141 inhibitors/antagomirs. The reporter activity was measured after 48 h. Mean and SDs are from 3 independent experiments *, P ≤ 0.05, t test. E, F, MDA-MB-231 cells stably expressing control-shRNA or PELP1-shRNA were used in these assays. Real-time qPCR (E) and Western blot analysis (F) of EMT gene expression in control shRNA, PELP1 shRNA model cells and PELP1 shRNA cells treated with miR inhibitors/antagomirs in MDA-MB-231 cells. *, P≤ 0.05; t test.
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Figure 2: MiRs influence the PELP1-mediated migratory and invasion potential. A, Boyden chamber analysis of the cell migration potential of the ZR cells transfected with mimetics and ZR-PELP1-shRNA cells transfected with inhibitors/antagomirs of miRs 200a and 141. B, MDA-MB-231 and MDA-MB-231-PELP1-shRNA cells were transfected with miRs 200a and 141 mimetics and inhibitors/antagomirs respectively and their effects on invasion were analyzed by using Matrigel invasion chamber assays. Mean and SDs are from three independent experiments. C, Western blot analysis of EMT genes expression in control, PELP1-shRNA cells and PELP1-shRNA clones treated with miR inhibitors/antagomirs. D, ZR-control-shRNA and ZR-PELP1-shRNA cells were transfected with ZEB1 and ZEB2 3′UTR reporter genes and treated with miR-200a and 141 inhibitors/antagomirs. The reporter activity was measured after 48 h. Mean and SDs are from 3 independent experiments *, P ≤ 0.05, t test. E, F, MDA-MB-231 cells stably expressing control-shRNA or PELP1-shRNA were used in these assays. Real-time qPCR (E) and Western blot analysis (F) of EMT gene expression in control shRNA, PELP1 shRNA model cells and PELP1 shRNA cells treated with miR inhibitors/antagomirs in MDA-MB-231 cells. *, P≤ 0.05; t test.

Mentions: Because miR-200 family members are implicated in metastasis and because PELP1 has the potential to regulate expression of miR200a/141, we tested the significance of miR-200a and miR-141 on PELP1-mediated migratory and invasion functions by using mimetics and antagomirs of miR-200a and miR-141. For these assays we have used established ZR75 (20) and MDA-MB-231 (18) model cells that stably express PELP1 shRNA. Stable expression of PELP1 shRNA reduced PELP1 expression to 70–80% of endogenous PELP1 in these models. As seen in previous published studies(17;21), PELP1 knockdown alter cell morphology, reduces motile F-actin containing structures in MDA-MB-231 cells. Similarly, PELP1 overexpression in less motile ZR-75 cells altered cell morphology and promoted F-actin containing motile structures (Supplementary Figure S2a, b). In migration assays using the Boyden chamber, PELP1 knockdown significantly reduced migration of ZR-75 cells compared to the migration of the control vector-transfected cells (Figure 2a). Similarly, mimetics of miR200a and miR141 also inhibited the migration of ZR75 cells. Conversely, treatment of ZR75-PELP1-shRNA cells with antagomirs of miR-200a and miR-141 rescued the defective migration seen in ZR75-PELP1-shRNA cells. To test the PELP1 effect on invasion, we used highly invasive MDA-MB-231 cells. PELP1 knockdown cells (MDA-MB-231-PELP1shRNA) had significantly reduced invasion compared to the invasion of the control cells (MDA-MB-231-conshRNA) (Figure 2b), while treatment of control MDA-MB-231 shRNA vector cells with the mimetics of miR200a/141 resulted in less invasion and treatment with antagomirs of miR-200a and miR-141 restored the lost invasion potential of the MDA-MB-231-PELP1shRNA cells. The results observed in ZR75 and MDA-MB-231-PELP1shRNA stable clones were also validated using transient knock down of PELP1 by siRNA (data not shown). Published studies implicate a role of miR-200 family members in the regulation of EMT genes (19). Since our earlier studies showed that PELP1 has the potential to regulate genes involved in EMT (18), we tested the hypothesis that PELP1 regulation of EMT genes may involve miR-200 family members. Western analysis revealed that miR-200a and miR-141 antagomirs reverses PELP1-shRNA mediated alterations in EMT genes in ER-positive cells (Figure 2c, Supplementary Figure S2c). Accordingly, in reporter gene-based assays PELP1-shRNA cells exhibited less ZEB1 and ZEB2 3′UTR luciferase reporter activity than the activity in control-shRNA cells and the PELP1 knockdown-mediated repression of reporter activity was relieved by the miR200a and miR141 antagomirs (Figure 2d). Similarly, miR-200a and miR-141 antagomirs also reversed PELP1-shRNA-mediated changes in EMT genes in ER-negative model cells (Figure 2e and f). Collectively, these results suggest that PELP1-mediated cell migratory/invasion functions involve the miR-200a and miR-141 family members.


Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Roy SS, Gonugunta VK, Bandyopadhyay A, Rao MK, Goodall GJ, Sun LZ, Tekmal RR, Vadlamudi RK - Oncogene (2013)

MiRs influence the PELP1-mediated migratory and invasion potential. A, Boyden chamber analysis of the cell migration potential of the ZR cells transfected with mimetics and ZR-PELP1-shRNA cells transfected with inhibitors/antagomirs of miRs 200a and 141. B, MDA-MB-231 and MDA-MB-231-PELP1-shRNA cells were transfected with miRs 200a and 141 mimetics and inhibitors/antagomirs respectively and their effects on invasion were analyzed by using Matrigel invasion chamber assays. Mean and SDs are from three independent experiments. C, Western blot analysis of EMT genes expression in control, PELP1-shRNA cells and PELP1-shRNA clones treated with miR inhibitors/antagomirs. D, ZR-control-shRNA and ZR-PELP1-shRNA cells were transfected with ZEB1 and ZEB2 3′UTR reporter genes and treated with miR-200a and 141 inhibitors/antagomirs. The reporter activity was measured after 48 h. Mean and SDs are from 3 independent experiments *, P ≤ 0.05, t test. E, F, MDA-MB-231 cells stably expressing control-shRNA or PELP1-shRNA were used in these assays. Real-time qPCR (E) and Western blot analysis (F) of EMT gene expression in control shRNA, PELP1 shRNA model cells and PELP1 shRNA cells treated with miR inhibitors/antagomirs in MDA-MB-231 cells. *, P≤ 0.05; t test.
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Figure 2: MiRs influence the PELP1-mediated migratory and invasion potential. A, Boyden chamber analysis of the cell migration potential of the ZR cells transfected with mimetics and ZR-PELP1-shRNA cells transfected with inhibitors/antagomirs of miRs 200a and 141. B, MDA-MB-231 and MDA-MB-231-PELP1-shRNA cells were transfected with miRs 200a and 141 mimetics and inhibitors/antagomirs respectively and their effects on invasion were analyzed by using Matrigel invasion chamber assays. Mean and SDs are from three independent experiments. C, Western blot analysis of EMT genes expression in control, PELP1-shRNA cells and PELP1-shRNA clones treated with miR inhibitors/antagomirs. D, ZR-control-shRNA and ZR-PELP1-shRNA cells were transfected with ZEB1 and ZEB2 3′UTR reporter genes and treated with miR-200a and 141 inhibitors/antagomirs. The reporter activity was measured after 48 h. Mean and SDs are from 3 independent experiments *, P ≤ 0.05, t test. E, F, MDA-MB-231 cells stably expressing control-shRNA or PELP1-shRNA were used in these assays. Real-time qPCR (E) and Western blot analysis (F) of EMT gene expression in control shRNA, PELP1 shRNA model cells and PELP1 shRNA cells treated with miR inhibitors/antagomirs in MDA-MB-231 cells. *, P≤ 0.05; t test.
Mentions: Because miR-200 family members are implicated in metastasis and because PELP1 has the potential to regulate expression of miR200a/141, we tested the significance of miR-200a and miR-141 on PELP1-mediated migratory and invasion functions by using mimetics and antagomirs of miR-200a and miR-141. For these assays we have used established ZR75 (20) and MDA-MB-231 (18) model cells that stably express PELP1 shRNA. Stable expression of PELP1 shRNA reduced PELP1 expression to 70–80% of endogenous PELP1 in these models. As seen in previous published studies(17;21), PELP1 knockdown alter cell morphology, reduces motile F-actin containing structures in MDA-MB-231 cells. Similarly, PELP1 overexpression in less motile ZR-75 cells altered cell morphology and promoted F-actin containing motile structures (Supplementary Figure S2a, b). In migration assays using the Boyden chamber, PELP1 knockdown significantly reduced migration of ZR-75 cells compared to the migration of the control vector-transfected cells (Figure 2a). Similarly, mimetics of miR200a and miR141 also inhibited the migration of ZR75 cells. Conversely, treatment of ZR75-PELP1-shRNA cells with antagomirs of miR-200a and miR-141 rescued the defective migration seen in ZR75-PELP1-shRNA cells. To test the PELP1 effect on invasion, we used highly invasive MDA-MB-231 cells. PELP1 knockdown cells (MDA-MB-231-PELP1shRNA) had significantly reduced invasion compared to the invasion of the control cells (MDA-MB-231-conshRNA) (Figure 2b), while treatment of control MDA-MB-231 shRNA vector cells with the mimetics of miR200a/141 resulted in less invasion and treatment with antagomirs of miR-200a and miR-141 restored the lost invasion potential of the MDA-MB-231-PELP1shRNA cells. The results observed in ZR75 and MDA-MB-231-PELP1shRNA stable clones were also validated using transient knock down of PELP1 by siRNA (data not shown). Published studies implicate a role of miR-200 family members in the regulation of EMT genes (19). Since our earlier studies showed that PELP1 has the potential to regulate genes involved in EMT (18), we tested the hypothesis that PELP1 regulation of EMT genes may involve miR-200 family members. Western analysis revealed that miR-200a and miR-141 antagomirs reverses PELP1-shRNA mediated alterations in EMT genes in ER-positive cells (Figure 2c, Supplementary Figure S2c). Accordingly, in reporter gene-based assays PELP1-shRNA cells exhibited less ZEB1 and ZEB2 3′UTR luciferase reporter activity than the activity in control-shRNA cells and the PELP1 knockdown-mediated repression of reporter activity was relieved by the miR200a and miR141 antagomirs (Figure 2d). Similarly, miR-200a and miR-141 antagomirs also reversed PELP1-shRNA-mediated changes in EMT genes in ER-negative model cells (Figure 2e and f). Collectively, these results suggest that PELP1-mediated cell migratory/invasion functions involve the miR-200a and miR-141 family members.

Bottom Line: Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2.Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis.Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, UT Health Science Center, San Antonio, TX, USA.

ABSTRACT
Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Show MeSH
Related in: MedlinePlus