In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated wit... more In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling. In this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing. Estrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment r...
Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechan... more Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to preven...
There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regard... more There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.
Journal of Pediatric Gastroenterology and Nutrition, 2012
Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli,... more Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/β-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/β-catenin signalling pathway. Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of β-catenin-expressing crypts by immunostaining was determined after 100-ng dickkopf treatment. Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P = 0.013) and 29% (P < 0.0001), crypt area to 42% (P = 0.0026) and 30% (P = 0.0067), and crypt fission to 51% (P = 0.006) and 29% (P < 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of β-catenin-expressing crypts decreased in dickkopf-treated animals. We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.
Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-cont... more Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-containing co-chaperones, such as small glutamine-rich tetratricopeptide repeat-containing alpha (SGTA). However, the precise mechanisms by which the predominantly cytoplasmic TPR proteins affect downstream transcriptional outcomes of steroid signaling remain unclear. In this study, we assessed how SGTA affects ligand sensitivity and action of the androgen receptor (AR) using a transactivation profiling approach. Deletion mapping coupled with structural prediction, transcriptional assays, and in vivo regulation of AR-responsive promoters were used to assess the role of SGTA domains in AR responses. At subsaturating ligand concentrations of %0 . 1 nM 5a-dihydrotestosterone, SGTA overexpression constricted AR activity by an average of 32% (P!0 . 002) across the majority of androgen-responsive loci tested, as well as on endogenous promoters in vivo. The strength of the SGTA effect was associated with the presence or absence of bioinformatically predicated transcription factor motifs at each site. Homodimerizaion of SGTA, which is thought to be necessary for chaperone complex formation, was found to be dependent on the structural integrity of amino acids 1-80, and a core evolutionary conserved peptide within this region (amino acids 21-40) necessary for an effect of SGTA on the activity of both exogenous and endogenous AR. This study provides new insights into the subdomain structure of SGTA and how SGTA acts as a regulator of AR ligand sensitivity. A change in AR:SGTA ratio will impact the cellular and molecular response of prostate cancer cells to maintain androgenic signals, which may influence tumor progression.
Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, su... more Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5adihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene-specific effects on basal or DHT-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100b levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.
Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and ... more Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3β-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-speci... more Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions, and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modelling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (c...
Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour... more Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro-tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma-associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERβ, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERβ transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen-driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4-dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface.
In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated wit... more In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling. In this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing. Estrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment r...
Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechan... more Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to preven...
There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regard... more There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.
Journal of Pediatric Gastroenterology and Nutrition, 2012
Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli,... more Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/β-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/β-catenin signalling pathway. Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of β-catenin-expressing crypts by immunostaining was determined after 100-ng dickkopf treatment. Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P = 0.013) and 29% (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001), crypt area to 42% (P = 0.0026) and 30% (P = 0.0067), and crypt fission to 51% (P = 0.006) and 29% (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of β-catenin-expressing crypts decreased in dickkopf-treated animals. We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.
Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-cont... more Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-containing co-chaperones, such as small glutamine-rich tetratricopeptide repeat-containing alpha (SGTA). However, the precise mechanisms by which the predominantly cytoplasmic TPR proteins affect downstream transcriptional outcomes of steroid signaling remain unclear. In this study, we assessed how SGTA affects ligand sensitivity and action of the androgen receptor (AR) using a transactivation profiling approach. Deletion mapping coupled with structural prediction, transcriptional assays, and in vivo regulation of AR-responsive promoters were used to assess the role of SGTA domains in AR responses. At subsaturating ligand concentrations of %0 . 1 nM 5a-dihydrotestosterone, SGTA overexpression constricted AR activity by an average of 32% (P!0 . 002) across the majority of androgen-responsive loci tested, as well as on endogenous promoters in vivo. The strength of the SGTA effect was associated with the presence or absence of bioinformatically predicated transcription factor motifs at each site. Homodimerizaion of SGTA, which is thought to be necessary for chaperone complex formation, was found to be dependent on the structural integrity of amino acids 1-80, and a core evolutionary conserved peptide within this region (amino acids 21-40) necessary for an effect of SGTA on the activity of both exogenous and endogenous AR. This study provides new insights into the subdomain structure of SGTA and how SGTA acts as a regulator of AR ligand sensitivity. A change in AR:SGTA ratio will impact the cellular and molecular response of prostate cancer cells to maintain androgenic signals, which may influence tumor progression.
Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, su... more Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5adihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene-specific effects on basal or DHT-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100b levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.
Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and ... more Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3β-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-speci... more Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions, and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modelling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (c...
Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour... more Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro-tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma-associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERβ, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERβ transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen-driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4-dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface.
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Papers by Andrew Trotta