INVESTIGADORES
ROSEMBLIT Cinthia
congresos y reuniones científicas
Título:
Progestin activation of p42/p44 MAPK induces Stat3 phosphorylation at serine 727 promoting breast cancer growth
Autor/es:
TKACH M; ROSEMBLIT C; BEGUELIN W; PROIETTI CJ; RIVAS MA; DIAZ FALQUE MC; CAYROL F; CHARREAU EH; ELIZALDE PV; SCHILLACI R
Lugar:
San Antonio, Texas
Reunión:
Congreso; San Antonio Breast Cancer Symposium; 2010
Institución organizadora:
American Association for Cancer Research (AACR)
Resumen:
We have previously demonstrated that the synthetic progestin medroxyprogesterone
acetate (MPA) induces Stat3 phosphorylation at Tyr705 leading to Stat3 transcriptional
activation, which is an absolute requirement for progestin stimulation of in vitro and in
vivo breast cancer growth. These studies were performed in C4HD cells from an
experimental model of hormonal carcinogenesis in which MPA induced mammary
adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Besides
tyrosine phosphorylation, Stat3 can also be phosphorylated on a single serine residue
(Ser727) leading to a full transcriptional activation of Stat3. Here, we explored the effect
of progestins on Stat3 Ser727 phosphorylation and its biological significance. MPA
treatment of C4HD and T47D cells for 5 to 10 min induced phosphorylation of Stat3 on
Ser727. The 727 Ser residue in Stat3 is situated in a conserved PMSP motive which
resembles the consensus PxS/TP motive for mitogen-activated protein kinase (MAPK)
targets. To address whether MPA activation of p42/p44 MAPK signaling pathway is
directly involved in Ser727 Stat3 phosphorylation, we pretreated C4HD cells with
U0126, a p42/p44 MAPK inhibitor, which suppressed phosphorylation of Stat3 on
Ser727. To strengthen the finding that Ser phosphorylation of Stat3 proceeds in a p42/p44
MAPK-dependent manner, we performed a cold in vitro phosphorylation assay. We
observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA
was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither
p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126
increased Stat3 Ser727 phosphorylation.
Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin
D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD
and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct
and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA
treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression
vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover,
MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in
cells transfected with Stat3S727-A. To further investigate the correlation between MPAinduced
Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were
transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A
had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells.
Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-in vitro and in
vivo breast cancer growth. These studies were performed in C4HD cells from an
experimental model of hormonal carcinogenesis in which MPA induced mammary
adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Besides
tyrosine phosphorylation, Stat3 can also be phosphorylated on a single serine residue
(Ser727) leading to a full transcriptional activation of Stat3. Here, we explored the effect
of progestins on Stat3 Ser727 phosphorylation and its biological significance. MPA
treatment of C4HD and T47D cells for 5 to 10 min induced phosphorylation of Stat3 on
Ser727. The 727 Ser residue in Stat3 is situated in a conserved PMSP motive which
resembles the consensus PxS/TP motive for mitogen-activated protein kinase (MAPK)
targets. To address whether MPA activation of p42/p44 MAPK signaling pathway is
directly involved in Ser727 Stat3 phosphorylation, we pretreated C4HD cells with
U0126, a p42/p44 MAPK inhibitor, which suppressed phosphorylation of Stat3 on
Ser727. To strengthen the finding that Ser phosphorylation of Stat3 proceeds in a p42/p44
MAPK-dependent manner, we performed a cold in vitro phosphorylation assay. We
observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA
was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither
p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126
increased Stat3 Ser727 phosphorylation.
Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin
D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD
and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct
and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA
treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression
vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover,
MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in
cells transfected with Stat3S727-A. To further investigate the correlation between MPAinduced
Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were
transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A
had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells.
Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-breast cancer growth. These studies were performed in C4HD cells from an
experimental model of hormonal carcinogenesis in which MPA induced mammary
adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Besides
tyrosine phosphorylation, Stat3 can also be phosphorylated on a single serine residue
(Ser727) leading to a full transcriptional activation of Stat3. Here, we explored the effect
of progestins on Stat3 Ser727 phosphorylation and its biological significance. MPA
treatment of C4HD and T47D cells for 5 to 10 min induced phosphorylation of Stat3 on
Ser727. The 727 Ser residue in Stat3 is situated in a conserved PMSP motive which
resembles the consensus PxS/TP motive for mitogen-activated protein kinase (MAPK)
targets. To address whether MPA activation of p42/p44 MAPK signaling pathway is
directly involved in Ser727 Stat3 phosphorylation, we pretreated C4HD cells with
U0126, a p42/p44 MAPK inhibitor, which suppressed phosphorylation of Stat3 on
Ser727. To strengthen the finding that Ser phosphorylation of Stat3 proceeds in a p42/p44
MAPK-dependent manner, we performed a cold in vitro phosphorylation assay. We
observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA
was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither
p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126
increased Stat3 Ser727 phosphorylation.
Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin
D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD
and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct
and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA
treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression
vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover,
MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in
cells transfected with Stat3S727-A. To further investigate the correlation between MPAinduced
Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were
transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A
had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells.
Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-in vitro phosphorylation assay. We
observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA
was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither
p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126
increased Stat3 Ser727 phosphorylation.
Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin
D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD
and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct
and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA
treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression
vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover,
MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in
cells transfected with Stat3S727-A. To further investigate the correlation between MPAinduced
Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were
transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A
had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells.
Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-in vitro MPA-induced growth of C4HD and T47D cells.
Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-in vivo progestin-driven breast
cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the
ability of these cells to grow in syngeneic mice. Our findings have for the first time
demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a
mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor
cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestininduced
cyclin D1 promoter activation and protein up-