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Estrogen receptor alpha prevents bladder cancer via INPP4B inhibited akt pathway in vitro and in vivo.

Hsu I, Yeh CR, Slavin S, Miyamoto H, Netto GJ, Tsai YC, Muyan M, Wu XR, Messing EM, Guancial EA, Yeh S - Oncotarget (2014)

Bottom Line: Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells.Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth.In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens.

View Article: PubMed Central - PubMed

Affiliation: Departments of Urology and Pathology, University of Rochester Medical Center, Rochester, NY 14642. Contributed equally.

ABSTRACT
Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre- carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.

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Total ERα knockout increased the cancer incidences in BBN-induced BCa model(A) ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of human Cytomegalovirus promoter (CMV-Cre) were bred with male floxERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (CMV-Cre/ERα−/+). Female CMV-Cre/ERα−/+ mice were then mated with male ERαfl/fl mice to generate WT and ERαKO mice. (B) Tail genomic DNA was isolated and mouse genotypes were identified by PCR using primers flanking ERα exon 3 and Cre. (C) mRNA was collected from whole bladders of female WT and ERαKO mice. Quantitative PCR (qPCR) was used to compare ERα mRNA level. **, p<0.01 compared to WT mice by t-test. (D) ERα expression was detected in bladders of female WT and ERαKO mice by IHC. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mice were shown at right (n=3 for each). ***, p<0.001 compared to WT mice by t-test. (E) Bladder weights were compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.0047 by t-test. (F) Representative images of BBN induced BCa of WT and ERαKO female mice at 35 weeks old. (G) BBN induced BCa incidence was compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.03 by Fisher's exact test.
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Figure 2: Total ERα knockout increased the cancer incidences in BBN-induced BCa model(A) ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of human Cytomegalovirus promoter (CMV-Cre) were bred with male floxERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (CMV-Cre/ERα−/+). Female CMV-Cre/ERα−/+ mice were then mated with male ERαfl/fl mice to generate WT and ERαKO mice. (B) Tail genomic DNA was isolated and mouse genotypes were identified by PCR using primers flanking ERα exon 3 and Cre. (C) mRNA was collected from whole bladders of female WT and ERαKO mice. Quantitative PCR (qPCR) was used to compare ERα mRNA level. **, p<0.01 compared to WT mice by t-test. (D) ERα expression was detected in bladders of female WT and ERαKO mice by IHC. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mice were shown at right (n=3 for each). ***, p<0.001 compared to WT mice by t-test. (E) Bladder weights were compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.0047 by t-test. (F) Representative images of BBN induced BCa of WT and ERαKO female mice at 35 weeks old. (G) BBN induced BCa incidence was compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.03 by Fisher's exact test.

Mentions: To investigate the in vivo role of ERα in BCa development, we employed cre-loxP strategy to knock out the floxed ERα gene (ERαKO) [34-36]. The breeding scheme for the generation of CMV-ERαKO (Fig. 2A) is presented with CMV-Cre mice crossed with floxed ERα. Genotyping results showed that CMV-ERαKO mice have both Cre and ERαKO alleles (Fig. 2B). Quantitative-PCR results show ERα mRNA from bladders of CMV-ERαKO mice was barely detectable compared to WT mice (Fig. 2C). IHC staining results further confirmed that ERα protein expression is ablated in CMV-ERαKO bladders (Fig. 2D).


Estrogen receptor alpha prevents bladder cancer via INPP4B inhibited akt pathway in vitro and in vivo.

Hsu I, Yeh CR, Slavin S, Miyamoto H, Netto GJ, Tsai YC, Muyan M, Wu XR, Messing EM, Guancial EA, Yeh S - Oncotarget (2014)

Total ERα knockout increased the cancer incidences in BBN-induced BCa model(A) ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of human Cytomegalovirus promoter (CMV-Cre) were bred with male floxERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (CMV-Cre/ERα−/+). Female CMV-Cre/ERα−/+ mice were then mated with male ERαfl/fl mice to generate WT and ERαKO mice. (B) Tail genomic DNA was isolated and mouse genotypes were identified by PCR using primers flanking ERα exon 3 and Cre. (C) mRNA was collected from whole bladders of female WT and ERαKO mice. Quantitative PCR (qPCR) was used to compare ERα mRNA level. **, p<0.01 compared to WT mice by t-test. (D) ERα expression was detected in bladders of female WT and ERαKO mice by IHC. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mice were shown at right (n=3 for each). ***, p<0.001 compared to WT mice by t-test. (E) Bladder weights were compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.0047 by t-test. (F) Representative images of BBN induced BCa of WT and ERαKO female mice at 35 weeks old. (G) BBN induced BCa incidence was compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.03 by Fisher's exact test.
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Figure 2: Total ERα knockout increased the cancer incidences in BBN-induced BCa model(A) ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of human Cytomegalovirus promoter (CMV-Cre) were bred with male floxERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (CMV-Cre/ERα−/+). Female CMV-Cre/ERα−/+ mice were then mated with male ERαfl/fl mice to generate WT and ERαKO mice. (B) Tail genomic DNA was isolated and mouse genotypes were identified by PCR using primers flanking ERα exon 3 and Cre. (C) mRNA was collected from whole bladders of female WT and ERαKO mice. Quantitative PCR (qPCR) was used to compare ERα mRNA level. **, p<0.01 compared to WT mice by t-test. (D) ERα expression was detected in bladders of female WT and ERαKO mice by IHC. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mice were shown at right (n=3 for each). ***, p<0.001 compared to WT mice by t-test. (E) Bladder weights were compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.0047 by t-test. (F) Representative images of BBN induced BCa of WT and ERαKO female mice at 35 weeks old. (G) BBN induced BCa incidence was compared between WT (n=28) and ERαKO (n=16) female mice at 35 weeks old. p=0.03 by Fisher's exact test.
Mentions: To investigate the in vivo role of ERα in BCa development, we employed cre-loxP strategy to knock out the floxed ERα gene (ERαKO) [34-36]. The breeding scheme for the generation of CMV-ERαKO (Fig. 2A) is presented with CMV-Cre mice crossed with floxed ERα. Genotyping results showed that CMV-ERαKO mice have both Cre and ERαKO alleles (Fig. 2B). Quantitative-PCR results show ERα mRNA from bladders of CMV-ERαKO mice was barely detectable compared to WT mice (Fig. 2C). IHC staining results further confirmed that ERα protein expression is ablated in CMV-ERαKO bladders (Fig. 2D).

Bottom Line: Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells.Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth.In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens.

View Article: PubMed Central - PubMed

Affiliation: Departments of Urology and Pathology, University of Rochester Medical Center, Rochester, NY 14642. Contributed equally.

ABSTRACT
Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre- carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.

Show MeSH
Related in: MedlinePlus