Limits...
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.

Show MeSH

Related in: MedlinePlus

Urothelial specific ERα gene knockout increased the cancer incidences in BBN induced BCa model(A) Urothelial ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of Uropleckin II promoter (UPII-Cre) were bred with male flox ERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (UPII-Cre/ERαfl/+). Male UPII-Cre/ERαfl/+ mice were then mated with female ERαfl/fl mice to generate WT and UPII-Cre/ERαKO mice. (B) Tail genomic DNA was isolated for genotyping by PCR using primers flanking ERα exon 3 and Cre primers. (C) ERα protein expression was detected in female bladders of WT and ERαKO mice by immunohistochemistry. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mouse bladder tissues were shown at right (n=4 for each) *, p<0.05 compared to WT mice by t-test. (D) Bladder weights were compared between WT (n=15) and UPII-ERαKO (n=9) female mice at 35 weeks old. p=0.0120 by t-test. (E) BBN induced BCa incidence was compared between WT (n=30) and UPII-ERαKO (n=21) female mice at 35 weeks old. p=0.0211 by Fisher's exact test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4202170&req=5

Figure 3: Urothelial specific ERα gene knockout increased the cancer incidences in BBN induced BCa model(A) Urothelial ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of Uropleckin II promoter (UPII-Cre) were bred with male flox ERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (UPII-Cre/ERαfl/+). Male UPII-Cre/ERαfl/+ mice were then mated with female ERαfl/fl mice to generate WT and UPII-Cre/ERαKO mice. (B) Tail genomic DNA was isolated for genotyping by PCR using primers flanking ERα exon 3 and Cre primers. (C) ERα protein expression was detected in female bladders of WT and ERαKO mice by immunohistochemistry. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mouse bladder tissues were shown at right (n=4 for each) *, p<0.05 compared to WT mice by t-test. (D) Bladder weights were compared between WT (n=15) and UPII-ERαKO (n=9) female mice at 35 weeks old. p=0.0120 by t-test. (E) BBN induced BCa incidence was compared between WT (n=30) and UPII-ERαKO (n=21) female mice at 35 weeks old. p=0.0211 by Fisher's exact test.

Mentions: In addition to CMV-ERαKO mice, we generated urothelium specific ERαKO mice (UPII-ERαKO) by breeding floxed ERα mice with UPII promoter driven Cre transgenic mice [39] (Fig. 3A). Genotyping results show that UPII-ERαKO mice have both Cre and floxed ERα alleles (Fig. 3B). The ERα KO allele cannot be detected from genotyping of DNA from tail snips of UPII-Cre driven knockout mice as Cre recombinase is only expressed in urothelial cells. IHC staining showed ERα protein is also ablated in UPII-ERαKO bladders (Fig. 3C). Female mice were fed with BBN water to induce tumors and then sacrificed at 35 weeks old. Higher bladder weight was found in the UPII-ERαKO mice than in WT mice (Fig. 3D) and histological analysis indicated that UPII-ERαKO mice have higher BCa incidence (76%) than WT mice (40%) (Fig. 3E), consistent with the phenotypes found in the CMV-ERαKO mice.


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)

Urothelial specific ERα gene knockout increased the cancer incidences in BBN induced BCa model(A) Urothelial ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of Uropleckin II promoter (UPII-Cre) were bred with male flox ERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (UPII-Cre/ERαfl/+). Male UPII-Cre/ERαfl/+ mice were then mated with female ERαfl/fl mice to generate WT and UPII-Cre/ERαKO mice. (B) Tail genomic DNA was isolated for genotyping by PCR using primers flanking ERα exon 3 and Cre primers. (C) ERα protein expression was detected in female bladders of WT and ERαKO mice by immunohistochemistry. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mouse bladder tissues were shown at right (n=4 for each) *, p<0.05 compared to WT mice by t-test. (D) Bladder weights were compared between WT (n=15) and UPII-ERαKO (n=9) female mice at 35 weeks old. p=0.0120 by t-test. (E) BBN induced BCa incidence was compared between WT (n=30) and UPII-ERαKO (n=21) female mice at 35 weeks old. p=0.0211 by Fisher's exact test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202170&req=5

Figure 3: Urothelial specific ERα gene knockout increased the cancer incidences in BBN induced BCa model(A) Urothelial ERαKO mouse breeding scheme. Female mice with Cre coding sequence under the control of Uropleckin II promoter (UPII-Cre) were bred with male flox ERα mice (ERαfl/fl) to generate heterozygous ERαKO mice (UPII-Cre/ERαfl/+). Male UPII-Cre/ERαfl/+ mice were then mated with female ERαfl/fl mice to generate WT and UPII-Cre/ERαKO mice. (B) Tail genomic DNA was isolated for genotyping by PCR using primers flanking ERα exon 3 and Cre primers. (C) ERα protein expression was detected in female bladders of WT and ERαKO mice by immunohistochemistry. Red arrows indicate cells expressing ERα protein. Quantifications of ERα positive cells in the mouse bladder tissues were shown at right (n=4 for each) *, p<0.05 compared to WT mice by t-test. (D) Bladder weights were compared between WT (n=15) and UPII-ERαKO (n=9) female mice at 35 weeks old. p=0.0120 by t-test. (E) BBN induced BCa incidence was compared between WT (n=30) and UPII-ERαKO (n=21) female mice at 35 weeks old. p=0.0211 by Fisher's exact test.
Mentions: In addition to CMV-ERαKO mice, we generated urothelium specific ERαKO mice (UPII-ERαKO) by breeding floxed ERα mice with UPII promoter driven Cre transgenic mice [39] (Fig. 3A). Genotyping results show that UPII-ERαKO mice have both Cre and floxed ERα alleles (Fig. 3B). The ERα KO allele cannot be detected from genotyping of DNA from tail snips of UPII-Cre driven knockout mice as Cre recombinase is only expressed in urothelial cells. IHC staining showed ERα protein is also ablated in UPII-ERαKO bladders (Fig. 3C). Female mice were fed with BBN water to induce tumors and then sacrificed at 35 weeks old. Higher bladder weight was found in the UPII-ERαKO mice than in WT mice (Fig. 3D) and histological analysis indicated that UPII-ERαKO mice have higher BCa incidence (76%) than WT mice (40%) (Fig. 3E), consistent with the phenotypes found in the CMV-ERαKO mice.

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