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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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PELP1-mediated epigenetic changes at miR-200 promoter involve HDAC2 and PELP1 interactions. A, ChIP assay was done using the DNA isolated from ZR and ZR-PELP1-shRNA cells with antibodies specific for HDAC2 or isotype rabbit IgG control. DNA recovered from the ChIP or input controls was subjected to real-time qPCR using primers that detect −156 to +83 and −236 to −107 promoter region of miR200b-200a-429 and miR200c-141 respectively. HDAC2 recruitment to promoter region of 200b-200a-429 and 200c-141 was determined. The promoter occupancy was calculated on the basis of the ratio of ChIP/input control. B, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200c-141 and miR200b-200a-429. After 48h the cells were treated with Trichostatin (TSA) for 24 h. Reporter activity was measured after 72 h. C, D, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200b-200a-429 (C) and miR200c-141 (D). After 24h the cells were transiently transfected with HDAC1, HDAC2, HDAC3 siRNAs. Reporter activity was measured after 72 h. E, F, Real-time qPCR analysis of miRs expression in ZR and ZR-PELP1 cells treated with Trichostatin (TSA) for 24 h (E) or HDAC2 siRNA for 72 h (F). The relative expression of each miR was quantified by measuring Ct values and normalizing against RNU19. *, P≤ 0.05; Student’s t test.
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Figure 4: PELP1-mediated epigenetic changes at miR-200 promoter involve HDAC2 and PELP1 interactions. A, ChIP assay was done using the DNA isolated from ZR and ZR-PELP1-shRNA cells with antibodies specific for HDAC2 or isotype rabbit IgG control. DNA recovered from the ChIP or input controls was subjected to real-time qPCR using primers that detect −156 to +83 and −236 to −107 promoter region of miR200b-200a-429 and miR200c-141 respectively. HDAC2 recruitment to promoter region of 200b-200a-429 and 200c-141 was determined. The promoter occupancy was calculated on the basis of the ratio of ChIP/input control. B, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200c-141 and miR200b-200a-429. After 48h the cells were treated with Trichostatin (TSA) for 24 h. Reporter activity was measured after 72 h. C, D, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200b-200a-429 (C) and miR200c-141 (D). After 24h the cells were transiently transfected with HDAC1, HDAC2, HDAC3 siRNAs. Reporter activity was measured after 72 h. E, F, Real-time qPCR analysis of miRs expression in ZR and ZR-PELP1 cells treated with Trichostatin (TSA) for 24 h (E) or HDAC2 siRNA for 72 h (F). The relative expression of each miR was quantified by measuring Ct values and normalizing against RNU19. *, P≤ 0.05; Student’s t test.

Mentions: Previous published findings established that PELP1 interacts with the histone deacetylase HDAC2 (25). Upregulation of H3K9Ac at the miR promoter region in PELP1 knockdown cells suggest a possibility that the PELP1 interaction with the HDAC2 enzyme may contribute to the alteration in acetylation levels at the miR-200 promoters. The ChIP assay with HDAC2 demonstrated the recruitment of HDAC2 at the regulatory regions of both miR promoters (Figure 4a). The role of PELP1 in HDAC2 recruitment at the miR200 promoters was further examined by performing a ChIP assay with HDAC2 in ZR-PELP1-shRNA cells (Figure 4a). Diminished recruitment of HDAC2 to the miR promoter regions was found in the absence of PELP1. Accordingly, the HDAC inhibitor Trichostatin A (TSA) treatment reversed PELP1-driven repressive effects on both miR200c-141 and miR200b-200a-429 promoter activities (Figure 4b). To further confirm the role and specificity of HDAC2 in PELP1 mediated regulation of miR-200a and miR-141, we have used HDAC2-siRNA. Non-targeting-, HDAC1- and HDAC3-siRNAs were used as additional controls and siRNA specificity was validated by Western blotting (Supplementary Figure 3d). The results showed that only HDAC2 siRNA but not HDAC1 or HDAC3 siRNA was able to reverse PELP1 mediated repression of miR-200a and miR-141 in miR promoter reporter assays (Figure 4c, d). Further, treatment with either TSA or HDAC2 siRNA was able to reverse PELP1 mediated repression of miR-200a and miR-141 (Figure 4e, f). These results suggest that PELP1 down regulates the expression of miR-200a and miR-141 by binding to and recruiting HDAC2 to their promoters.


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)

PELP1-mediated epigenetic changes at miR-200 promoter involve HDAC2 and PELP1 interactions. A, ChIP assay was done using the DNA isolated from ZR and ZR-PELP1-shRNA cells with antibodies specific for HDAC2 or isotype rabbit IgG control. DNA recovered from the ChIP or input controls was subjected to real-time qPCR using primers that detect −156 to +83 and −236 to −107 promoter region of miR200b-200a-429 and miR200c-141 respectively. HDAC2 recruitment to promoter region of 200b-200a-429 and 200c-141 was determined. The promoter occupancy was calculated on the basis of the ratio of ChIP/input control. B, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200c-141 and miR200b-200a-429. After 48h the cells were treated with Trichostatin (TSA) for 24 h. Reporter activity was measured after 72 h. C, D, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200b-200a-429 (C) and miR200c-141 (D). After 24h the cells were transiently transfected with HDAC1, HDAC2, HDAC3 siRNAs. Reporter activity was measured after 72 h. E, F, Real-time qPCR analysis of miRs expression in ZR and ZR-PELP1 cells treated with Trichostatin (TSA) for 24 h (E) or HDAC2 siRNA for 72 h (F). The relative expression of each miR was quantified by measuring Ct values and normalizing against RNU19. *, P≤ 0.05; Student’s t test.
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Figure 4: PELP1-mediated epigenetic changes at miR-200 promoter involve HDAC2 and PELP1 interactions. A, ChIP assay was done using the DNA isolated from ZR and ZR-PELP1-shRNA cells with antibodies specific for HDAC2 or isotype rabbit IgG control. DNA recovered from the ChIP or input controls was subjected to real-time qPCR using primers that detect −156 to +83 and −236 to −107 promoter region of miR200b-200a-429 and miR200c-141 respectively. HDAC2 recruitment to promoter region of 200b-200a-429 and 200c-141 was determined. The promoter occupancy was calculated on the basis of the ratio of ChIP/input control. B, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200c-141 and miR200b-200a-429. After 48h the cells were treated with Trichostatin (TSA) for 24 h. Reporter activity was measured after 72 h. C, D, ZR and ZR-PELP1 cells were transfected with Luc-promoter reporters of miR200b-200a-429 (C) and miR200c-141 (D). After 24h the cells were transiently transfected with HDAC1, HDAC2, HDAC3 siRNAs. Reporter activity was measured after 72 h. E, F, Real-time qPCR analysis of miRs expression in ZR and ZR-PELP1 cells treated with Trichostatin (TSA) for 24 h (E) or HDAC2 siRNA for 72 h (F). The relative expression of each miR was quantified by measuring Ct values and normalizing against RNU19. *, P≤ 0.05; Student’s t test.
Mentions: Previous published findings established that PELP1 interacts with the histone deacetylase HDAC2 (25). Upregulation of H3K9Ac at the miR promoter region in PELP1 knockdown cells suggest a possibility that the PELP1 interaction with the HDAC2 enzyme may contribute to the alteration in acetylation levels at the miR-200 promoters. The ChIP assay with HDAC2 demonstrated the recruitment of HDAC2 at the regulatory regions of both miR promoters (Figure 4a). The role of PELP1 in HDAC2 recruitment at the miR200 promoters was further examined by performing a ChIP assay with HDAC2 in ZR-PELP1-shRNA cells (Figure 4a). Diminished recruitment of HDAC2 to the miR promoter regions was found in the absence of PELP1. Accordingly, the HDAC inhibitor Trichostatin A (TSA) treatment reversed PELP1-driven repressive effects on both miR200c-141 and miR200b-200a-429 promoter activities (Figure 4b). To further confirm the role and specificity of HDAC2 in PELP1 mediated regulation of miR-200a and miR-141, we have used HDAC2-siRNA. Non-targeting-, HDAC1- and HDAC3-siRNAs were used as additional controls and siRNA specificity was validated by Western blotting (Supplementary Figure 3d). The results showed that only HDAC2 siRNA but not HDAC1 or HDAC3 siRNA was able to reverse PELP1 mediated repression of miR-200a and miR-141 in miR promoter reporter assays (Figure 4c, d). Further, treatment with either TSA or HDAC2 siRNA was able to reverse PELP1 mediated repression of miR-200a and miR-141 (Figure 4e, f). These results suggest that PELP1 down regulates the expression of miR-200a and miR-141 by binding to and recruiting HDAC2 to their promoters.

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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Related in: MedlinePlus