Transforming growth factor-!1 (TGF-!1) is a pluripotent
cytokine that can inhibit epithelial prol... more Transforming growth factor-!1 (TGF-!1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-!1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-!. Activation was highly restricted. TGF-!1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-!1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-!1 immunoreactivity, which suggests that TGF-!1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-!1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-!1 activity, we manipulated TGF-!1 levels in vivo using Tgf!1 knockout mice and undertook tissue recombination experiments with heterozygous tissue.In Tgf!1 heterozygous mice, which have <10% wildtype levels of TGF-!1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgf!1"/# epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-!1 was critical to the proliferative phenotype, Tgf!1"/" and "/# epithelium were transplanted into "/" mammary stroma. The outgrowth of Tgf!1"/# epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgf!1"/# than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-!1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-!1 activation, which in turn restricts proliferative response to hormone signaling.
Transforming growth factor (TGF)-beta1 is a potent inhibitor
of mammary epithelial proliferation.... more Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely colocalize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptorphosphorylated Smad 2/3, indicating that TGF-betasignaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgfbeta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.
Valproic acid (VPA) is an effective antiepileptic drug with an additional activity for the treatm... more Valproic acid (VPA) is an effective antiepileptic drug with an additional activity for the treatment of bipolar disorder. It has been assumed that both activities arise from a common target. At the molecular level, VPA targets a number of distinct proteins that are involved in signal transduction. VPA inhibition of inositol synthase reduces the cellular concentration of myo-inositol, an effect common to the mood stabilizers lithium and carbamazepine. VPA inhibition of histone deacetylases activates Wnt signaling via elevated -catenin expression and causes teratogenicity. Given the VPA chemical structure, it may be possible to design VPA derivatives and analogs that modulate specific protein targets but leave the others unaffected. Indeed, it has been shown that some nonteratogenic VPA derivatives retain antiepileptic and inositol signaling effects. In this A.J.H. and E.C.D. are supported by Wellcome Trust Program grant 07450. Article, publication date, and citation information can be found at
The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding ... more The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, β-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of β-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wntdependent tumors.
Transforming growth factor-!1 (TGF-!1) is a pluripotent
cytokine that can inhibit epithelial prol... more Transforming growth factor-!1 (TGF-!1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-!1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-!. Activation was highly restricted. TGF-!1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-!1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-!1 immunoreactivity, which suggests that TGF-!1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-!1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-!1 activity, we manipulated TGF-!1 levels in vivo using Tgf!1 knockout mice and undertook tissue recombination experiments with heterozygous tissue.In Tgf!1 heterozygous mice, which have <10% wildtype levels of TGF-!1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgf!1"/# epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-!1 was critical to the proliferative phenotype, Tgf!1"/" and "/# epithelium were transplanted into "/" mammary stroma. The outgrowth of Tgf!1"/# epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgf!1"/# than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-!1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-!1 activation, which in turn restricts proliferative response to hormone signaling.
Transforming growth factor (TGF)-beta1 is a potent inhibitor
of mammary epithelial proliferation.... more Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely colocalize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptorphosphorylated Smad 2/3, indicating that TGF-betasignaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgfbeta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.
Valproic acid (VPA) is an effective antiepileptic drug with an additional activity for the treatm... more Valproic acid (VPA) is an effective antiepileptic drug with an additional activity for the treatment of bipolar disorder. It has been assumed that both activities arise from a common target. At the molecular level, VPA targets a number of distinct proteins that are involved in signal transduction. VPA inhibition of inositol synthase reduces the cellular concentration of myo-inositol, an effect common to the mood stabilizers lithium and carbamazepine. VPA inhibition of histone deacetylases activates Wnt signaling via elevated -catenin expression and causes teratogenicity. Given the VPA chemical structure, it may be possible to design VPA derivatives and analogs that modulate specific protein targets but leave the others unaffected. Indeed, it has been shown that some nonteratogenic VPA derivatives retain antiepileptic and inositol signaling effects. In this A.J.H. and E.C.D. are supported by Wellcome Trust Program grant 07450. Article, publication date, and citation information can be found at
The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding ... more The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, β-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of β-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wntdependent tumors.
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Papers by K. Ewan
cytokine that can inhibit epithelial proliferation
and induce apoptosis, but is also widely implicated in
breast cancer progression. Understanding its biological
action in mammary development is critical for
understanding its role in cancer. TGF-!1 is produced
as a latent complex that requires extracellular activation
before receptor binding. To better understand
the spatial and temporal regulation of its action during
mammary gland development, we examined the
pattern of activation in situ using antibodies selected
to distinguish between latent and active TGF-!. Activation
was highly restricted. TGF-!1 activation was
localized primarily to the epithelium, and within the
epithelium it was restricted to luminal epithelial cells
but absent from either cap or myoepithelial cells.
Within the luminal epithelium, we noted a further
restriction. During periods of proliferation (ie, puberty,
estrus and pregnancy), which are stimulated
by ovarian hormones, TGF-!1 activation decreased in
some cells, consistent with preparation for proliferation.
Paradoxically, other cells simultaneously increase
TGF-!1 immunoreactivity, which suggests that
TGF-!1 differentially restrains epithelial subpopulations
from responding to hormonal signals to proliferate.
These data suggest that endogenous TGF-!1 activation
and thus activity are regulated by ovarian
hormones. To determine the specific consequences of
TGF-!1 activity, we manipulated TGF-!1 levels in vivo
using Tgf!1 knockout mice and undertook tissue recombination
experiments with heterozygous tissue.In Tgf!1 heterozygous mice, which have <10% wildtype
levels of TGF-!1, ductal development during puberty
and alveolar development during pregnancy
were accelerated, consistent with its role as a growth
inhibitor. The proliferative index of Tgf!1"/# epithelium
was increased approximately twofold in quiescent
tissue and fourfold in proliferating tissue but
both ducts and alveoli were grossly and histologically
normal. To test whether epithelial TGF-!1 was critical
to the proliferative phenotype, Tgf!1"/" and "/#
epithelium were transplanted into "/" mammary
stroma. The outgrowth of Tgf!1"/# epithelium was
accelerated in wild-type hosts, indicating that the
phenotype was intrinsic to the epithelium. Moreover,
proliferation was 15-fold greater in Tgf!1"/# than
wild-type mice after ovariectomy and treatment with
estrogen and progesterone, suggesting that TGF-!1
acts in an autocrine or juxtacrine manner to regulate
epithelial proliferation. Together these data indicate
that ovarian hormones regulate TGF-!1 activation,
which in turn restricts proliferative response to hormone
signaling.
of mammary epithelial proliferation. In human
breast, estrogen receptor (ER)-alpha cells rarely colocalize
with markers of proliferation, but their
increased frequency correlates with breast cancer
risk. To determine whether TGF-beta1 is necessary for
the quiescence of ER-alpha-positive populations, we examined
mouse mammary epithelial glands at estrus.
Approximately 35% of epithelial cells showed TGF-beta1
activation, which co-localized with nuclear receptorphosphorylated
Smad 2/3, indicating that TGF-betasignaling
is autocrine. Nuclear Smad co-localized with
nuclear ER-alpha. To test whether TGF-beta inhibits proliferation,
we examined genetically engineered mice with
different levels of TGF-beta1. ER-alpha co-localization with
markers of proliferation (ie, Ki-67 or bromodeoxyuridine)
at estrus was significantly increased in the
mammary glands of Tgfbeta1 C57/bl/129SV heterozygote
mice. This relationship was maintained after
pregnancy but was absent at puberty. Conversely,
mammary epithelial expression of constitutively active
TGF-beta1 via the MMTV promoter suppressed proliferation
of ER-alpha-positive cells. Thus, TGF-beta1 activation
functionally restrains ER-alpha-positive cells from
proliferating in adult mammary gland. Accordingly,
we propose that TGF-beta1 dysregulation may promote
proliferation of ER-alpha-positive cells associated with
breast cancer risk in humans.
cytokine that can inhibit epithelial proliferation
and induce apoptosis, but is also widely implicated in
breast cancer progression. Understanding its biological
action in mammary development is critical for
understanding its role in cancer. TGF-!1 is produced
as a latent complex that requires extracellular activation
before receptor binding. To better understand
the spatial and temporal regulation of its action during
mammary gland development, we examined the
pattern of activation in situ using antibodies selected
to distinguish between latent and active TGF-!. Activation
was highly restricted. TGF-!1 activation was
localized primarily to the epithelium, and within the
epithelium it was restricted to luminal epithelial cells
but absent from either cap or myoepithelial cells.
Within the luminal epithelium, we noted a further
restriction. During periods of proliferation (ie, puberty,
estrus and pregnancy), which are stimulated
by ovarian hormones, TGF-!1 activation decreased in
some cells, consistent with preparation for proliferation.
Paradoxically, other cells simultaneously increase
TGF-!1 immunoreactivity, which suggests that
TGF-!1 differentially restrains epithelial subpopulations
from responding to hormonal signals to proliferate.
These data suggest that endogenous TGF-!1 activation
and thus activity are regulated by ovarian
hormones. To determine the specific consequences of
TGF-!1 activity, we manipulated TGF-!1 levels in vivo
using Tgf!1 knockout mice and undertook tissue recombination
experiments with heterozygous tissue.In Tgf!1 heterozygous mice, which have <10% wildtype
levels of TGF-!1, ductal development during puberty
and alveolar development during pregnancy
were accelerated, consistent with its role as a growth
inhibitor. The proliferative index of Tgf!1"/# epithelium
was increased approximately twofold in quiescent
tissue and fourfold in proliferating tissue but
both ducts and alveoli were grossly and histologically
normal. To test whether epithelial TGF-!1 was critical
to the proliferative phenotype, Tgf!1"/" and "/#
epithelium were transplanted into "/" mammary
stroma. The outgrowth of Tgf!1"/# epithelium was
accelerated in wild-type hosts, indicating that the
phenotype was intrinsic to the epithelium. Moreover,
proliferation was 15-fold greater in Tgf!1"/# than
wild-type mice after ovariectomy and treatment with
estrogen and progesterone, suggesting that TGF-!1
acts in an autocrine or juxtacrine manner to regulate
epithelial proliferation. Together these data indicate
that ovarian hormones regulate TGF-!1 activation,
which in turn restricts proliferative response to hormone
signaling.
of mammary epithelial proliferation. In human
breast, estrogen receptor (ER)-alpha cells rarely colocalize
with markers of proliferation, but their
increased frequency correlates with breast cancer
risk. To determine whether TGF-beta1 is necessary for
the quiescence of ER-alpha-positive populations, we examined
mouse mammary epithelial glands at estrus.
Approximately 35% of epithelial cells showed TGF-beta1
activation, which co-localized with nuclear receptorphosphorylated
Smad 2/3, indicating that TGF-betasignaling
is autocrine. Nuclear Smad co-localized with
nuclear ER-alpha. To test whether TGF-beta inhibits proliferation,
we examined genetically engineered mice with
different levels of TGF-beta1. ER-alpha co-localization with
markers of proliferation (ie, Ki-67 or bromodeoxyuridine)
at estrus was significantly increased in the
mammary glands of Tgfbeta1 C57/bl/129SV heterozygote
mice. This relationship was maintained after
pregnancy but was absent at puberty. Conversely,
mammary epithelial expression of constitutively active
TGF-beta1 via the MMTV promoter suppressed proliferation
of ER-alpha-positive cells. Thus, TGF-beta1 activation
functionally restrains ER-alpha-positive cells from
proliferating in adult mammary gland. Accordingly,
we propose that TGF-beta1 dysregulation may promote
proliferation of ER-alpha-positive cells associated with
breast cancer risk in humans.