CITED1 is a non-DNA binding transcriptional co-regulator whose expression can 29 distinguish the ... more CITED1 is a non-DNA binding transcriptional co-regulator whose expression can 29 distinguish the 'proliferative' from 'invasive' signature in the phenotype-switching 30 model of melanoma. We have found that CITED1 expression is repressed by TGFβ in 31 addition to other 'proliferative' signature genes while the 'invasive' signature genes 32 are upregulated. In agreement, CITED1 positively correlates with MITF expression 33 and can discriminate the MITF-high/pigmentation tumor molecular subtype in a 34 large cohort (120) of melanoma cell lines. Interestingly, CITED1 overexpression 35 significantly suppressed MITF promoter activation, mRNA and protein expression 36 levels while MITF was transiently upregulated following siRNA mediated CITED1 37 silencing. Conversely, MITF siRNA silencing resulted in CITED1 downregulation 38 indicating a reciprocal relationship. Whole genome expression analysis identified a 39 phenotype shift induced by CITED1 silencing and driven mainly by expression of MITF 40 and a cohort of MITF target genes that were significantly altered. Concomitantly, we 41 found changes in the cell-cycle profile that manifest as transient G1 accumulation, 42 increased expression of CDKN1A and a reduction in cell viability. Additionally, we 43 could predict survival outcome by classifying primary melanoma tumors using our in 44 vitro derived 'CITED1-silenced' gene expression signature. We hypothesize that 45 CITED1 acts a regulator of MITF, functioning to maintain MITF levels in a range 46 compatible with tumourigenesis.
PrePrints sought to explain the observation that melanoma cells altered between two states: those... more PrePrints sought to explain the observation that melanoma cells altered between two states: those with high proliferative potential that are less invasive and those with high metastatic potential that are less proliferative. These separate but alternating states are controlled by different transcriptional programs and can be defined by specific gene signatures (Hoek, Eichhoff, et al. 2008a). MITF expression and many of its known targets (TYR, MLANA) define the 'proliferative' group, while the 'invasive' signature group is characterized by expression of negative regulators of the Wnt signaling pathway (WNT5A, DKK1, CTGF). CITED1 expression was associated with the proliferative pathway signature and subsequently confirmed in an updated and expanded data set to be significantly correlated with the proliferative phenotype (P<1.00E-05, http://www.jurmo.ch/hopp, accessed 19 March 2013) (Hoek et al. 2006) , (Widmer et al. 2012). Studies on CITED1 suggest that it is a non-DNA binding nuclear transcriptional co-regulator capable of influencing TGFβ induced transcription mediated by ligand-induced SMAD hetero-oligomerization; estrogendependent transcription mediated by ERα, and Wnt/β-Catenin-dependent transcription. These effects are dependent on CITED1-CBP/P300 binding via the conserved CITED family CR2 domain and while in the case of ERα, CITED1
Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) media... more Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well‐characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal‐like, luminal A, luminal B, HER2‐enriched and normal‐like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2−/Ki67low, ER+/HER2−/Ki67high and HER2+), and were val...
PrePrints sought to explain the observation that melanoma cells altered between two states: those... more PrePrints sought to explain the observation that melanoma cells altered between two states: those with high proliferative potential that are less invasive and those with high metastatic potential that are less proliferative. These separate but alternating states are controlled by different transcriptional programs and can be defined by specific gene signatures (Hoek, Eichhoff, et al. 2008a). MITF expression and many of its known targets (TYR, MLANA) define the 'proliferative' group, while the 'invasive' signature group is characterized by expression of negative regulators of the Wnt signaling pathway (WNT5A, DKK1, CTGF). CITED1 expression was associated with the proliferative pathway signature and subsequently confirmed in an updated and expanded data set to be significantly correlated with the proliferative phenotype (P<1.00E-05, http://www.jurmo.ch/hopp, accessed 19 March 2013) (Hoek et al. 2006) , (Widmer et al. 2012). Studies on CITED1 suggest that it is a non-DNA binding nuclear transcriptional co-regulator capable of influencing TGFβ induced transcription mediated by ligand-induced SMAD hetero-oligomerization; estrogendependent transcription mediated by ERα, and Wnt/β-Catenin-dependent transcription. These effects are dependent on CITED1-CBP/P300 binding via the conserved CITED family CR2 domain and while in the case of ERα, CITED1
Journal of Mammary Gland Biology and Neoplasia, 2008
Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that genera... more Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that generates the mammary tree that will eventually fill the fat pad. This estrogen-dependent process is believed to be essentially dependent on locally produced growth factors that act in a paracrine fashion. EGF-like growth factor ligands, acting through EGF receptors are some of the principal promoters of pubertal ductal morphogenesis. Amphiregulin is the most abundant EGF-like growth factor in the pubertal mammary gland. Its gene is transcriptionally regulated by ERα, and recent evidence identifies it as a key mediator of the estrogen-driven epithelial cell proliferation of puberty: The pubertal deficiency in mammary gland ductal morphogenesis in ERα, amphiregulin, and EGFR knockout mice phenocopy each other. As a prognostic indicator in human breast cancer, amphiregulin indicates an outcome identical to that predicted by ERα presence. Despite this, a range of studies both on preneoplastic human breast tissue and on cell culture based models of breast cancer, suggest a possibly significant role for amphiregulin in driving human breast cancer progression. Here we summarise our current understanding of amphiregulin’s contribution to mammary gland development and breast cancer progression.
Proceedings of The National Academy of Sciences, 2009
The influential role of Wnt5a in tumor progression underscores the requirement for developing mol... more The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5amediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGF1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGF1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGF1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly.
Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased drama... more Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets. (Part of a Multi-author Review)
The cytokine tumor necrosis factor alpha (TNF-K K) is a critical e¡ector of the pathogenesis of r... more The cytokine tumor necrosis factor alpha (TNF-K K) is a critical e¡ector of the pathogenesis of rheumatoid arthritis (RA). We used oligonucleotide microarray (OM) analysis to assess TNF-K K-modulated gene expression in cultured primary human synoviocytes in vitro. Genes identi¢ed include cytokines and in£ammatory mediators, extracellular matrix and adhesion molecules, cell cycle and proliferation related proteins, transcription related proteins, and apoptotic mediators. OM identi-¢ed 1185 di¡erentially expressed genes in TNF-K K-treated synoviocytes. The regulation of Nef-associated factor-1 (Naf1), an A20-binding, nuclear factor kappa B (NFU UB) inhibitory protein was probed further given its putative role as an endogenous brake for the expression of some TNF-K K-driven genes. Naf1 mRNA levels were higher in synovial biopsies from patients with active RA and seronegative arthropathy than in those from patients with osteoarthritis. These ¢ndings underscore the value of transcriptome analysis in cytokine-activated synoviocyte cultures in vitro as a means of identifying disease-associated genes in human arthritis, and implicate Naf1 as a potential modulator of TNF-K K bioactivity in RA. ß
Tumor cell migration plays a central role in the process of cancer metastasis. We recently identi... more Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 receptor or Galpha(s) expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr-34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Galpha(s)/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.
Journal of mammary gland biology and neoplasia, 2006
During puberty the mammary gland develops from a rudimentary tree to a branched epithelial networ... more During puberty the mammary gland develops from a rudimentary tree to a branched epithelial network of ducts which can support alveolar development and subsequent milk production during pregnancy and lactation. This process involves growth, proliferation, migration, branching, invasion, apoptosis and above all, tight regulation which allows these processes to take place simultaneously during the course of just a few weeks to create an adult gland. The process is under hormonal control and is thus coordinated with reproductive development. Mouse models, with overexpressed or knocked-out genes, have highlighted a number of pubertal mammary gland phenotypes and given significant insight into the regulatory mechanisms controlling this period of development. Here we review the published findings of the wide range of gene-manipulated mammary mouse models, documenting the common pubertal mammary gland phenotypes observed, and summarizing their contribution to our current understanding of how pubertal mammary gland development occurs.
Journal of the American Society of Nephrology : JASN, 2008
Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by h... more Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.
Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In... more Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells -but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation. In the present study we sought to investigate whether negative targeting of TNK2 by siRNA could be used to inhibit cancer cell invasion, to establish the contribution of its effect on the EGFR and to consequently attempt to resolve the issue of TNK2's mechanism of action.
Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased drama... more Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets. (Part of a Multi-author Review)
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse ma... more Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-b and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERa binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERa localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERa. Similar to ERa, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERa-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Abstract The influential role of Wnt5a in tumor progression underscores the requirement for devel... more Abstract The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63.
Abstract. Utilisation of omics technologies, in particular gene expression profiling, has increas... more Abstract. Utilisation of omics technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, highthroughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers.
Abstract Expression microarray analysis identified over 930 genes regulated during puberty in the... more Abstract Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented.
Abstract Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prog... more Abstract Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells–but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation.
Abstract Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregul... more Abstract Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects.
CITED1 is a non-DNA binding transcriptional co-regulator whose expression can 29 distinguish the ... more CITED1 is a non-DNA binding transcriptional co-regulator whose expression can 29 distinguish the 'proliferative' from 'invasive' signature in the phenotype-switching 30 model of melanoma. We have found that CITED1 expression is repressed by TGFβ in 31 addition to other 'proliferative' signature genes while the 'invasive' signature genes 32 are upregulated. In agreement, CITED1 positively correlates with MITF expression 33 and can discriminate the MITF-high/pigmentation tumor molecular subtype in a 34 large cohort (120) of melanoma cell lines. Interestingly, CITED1 overexpression 35 significantly suppressed MITF promoter activation, mRNA and protein expression 36 levels while MITF was transiently upregulated following siRNA mediated CITED1 37 silencing. Conversely, MITF siRNA silencing resulted in CITED1 downregulation 38 indicating a reciprocal relationship. Whole genome expression analysis identified a 39 phenotype shift induced by CITED1 silencing and driven mainly by expression of MITF 40 and a cohort of MITF target genes that were significantly altered. Concomitantly, we 41 found changes in the cell-cycle profile that manifest as transient G1 accumulation, 42 increased expression of CDKN1A and a reduction in cell viability. Additionally, we 43 could predict survival outcome by classifying primary melanoma tumors using our in 44 vitro derived 'CITED1-silenced' gene expression signature. We hypothesize that 45 CITED1 acts a regulator of MITF, functioning to maintain MITF levels in a range 46 compatible with tumourigenesis.
PrePrints sought to explain the observation that melanoma cells altered between two states: those... more PrePrints sought to explain the observation that melanoma cells altered between two states: those with high proliferative potential that are less invasive and those with high metastatic potential that are less proliferative. These separate but alternating states are controlled by different transcriptional programs and can be defined by specific gene signatures (Hoek, Eichhoff, et al. 2008a). MITF expression and many of its known targets (TYR, MLANA) define the 'proliferative' group, while the 'invasive' signature group is characterized by expression of negative regulators of the Wnt signaling pathway (WNT5A, DKK1, CTGF). CITED1 expression was associated with the proliferative pathway signature and subsequently confirmed in an updated and expanded data set to be significantly correlated with the proliferative phenotype (P<1.00E-05, http://www.jurmo.ch/hopp, accessed 19 March 2013) (Hoek et al. 2006) , (Widmer et al. 2012). Studies on CITED1 suggest that it is a non-DNA binding nuclear transcriptional co-regulator capable of influencing TGFβ induced transcription mediated by ligand-induced SMAD hetero-oligomerization; estrogendependent transcription mediated by ERα, and Wnt/β-Catenin-dependent transcription. These effects are dependent on CITED1-CBP/P300 binding via the conserved CITED family CR2 domain and while in the case of ERα, CITED1
Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) media... more Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well‐characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal‐like, luminal A, luminal B, HER2‐enriched and normal‐like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2−/Ki67low, ER+/HER2−/Ki67high and HER2+), and were val...
PrePrints sought to explain the observation that melanoma cells altered between two states: those... more PrePrints sought to explain the observation that melanoma cells altered between two states: those with high proliferative potential that are less invasive and those with high metastatic potential that are less proliferative. These separate but alternating states are controlled by different transcriptional programs and can be defined by specific gene signatures (Hoek, Eichhoff, et al. 2008a). MITF expression and many of its known targets (TYR, MLANA) define the 'proliferative' group, while the 'invasive' signature group is characterized by expression of negative regulators of the Wnt signaling pathway (WNT5A, DKK1, CTGF). CITED1 expression was associated with the proliferative pathway signature and subsequently confirmed in an updated and expanded data set to be significantly correlated with the proliferative phenotype (P<1.00E-05, http://www.jurmo.ch/hopp, accessed 19 March 2013) (Hoek et al. 2006) , (Widmer et al. 2012). Studies on CITED1 suggest that it is a non-DNA binding nuclear transcriptional co-regulator capable of influencing TGFβ induced transcription mediated by ligand-induced SMAD hetero-oligomerization; estrogendependent transcription mediated by ERα, and Wnt/β-Catenin-dependent transcription. These effects are dependent on CITED1-CBP/P300 binding via the conserved CITED family CR2 domain and while in the case of ERα, CITED1
Journal of Mammary Gland Biology and Neoplasia, 2008
Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that genera... more Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that generates the mammary tree that will eventually fill the fat pad. This estrogen-dependent process is believed to be essentially dependent on locally produced growth factors that act in a paracrine fashion. EGF-like growth factor ligands, acting through EGF receptors are some of the principal promoters of pubertal ductal morphogenesis. Amphiregulin is the most abundant EGF-like growth factor in the pubertal mammary gland. Its gene is transcriptionally regulated by ERα, and recent evidence identifies it as a key mediator of the estrogen-driven epithelial cell proliferation of puberty: The pubertal deficiency in mammary gland ductal morphogenesis in ERα, amphiregulin, and EGFR knockout mice phenocopy each other. As a prognostic indicator in human breast cancer, amphiregulin indicates an outcome identical to that predicted by ERα presence. Despite this, a range of studies both on preneoplastic human breast tissue and on cell culture based models of breast cancer, suggest a possibly significant role for amphiregulin in driving human breast cancer progression. Here we summarise our current understanding of amphiregulin’s contribution to mammary gland development and breast cancer progression.
Proceedings of The National Academy of Sciences, 2009
The influential role of Wnt5a in tumor progression underscores the requirement for developing mol... more The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5amediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGF1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGF1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGF1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly.
Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased drama... more Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets. (Part of a Multi-author Review)
The cytokine tumor necrosis factor alpha (TNF-K K) is a critical e¡ector of the pathogenesis of r... more The cytokine tumor necrosis factor alpha (TNF-K K) is a critical e¡ector of the pathogenesis of rheumatoid arthritis (RA). We used oligonucleotide microarray (OM) analysis to assess TNF-K K-modulated gene expression in cultured primary human synoviocytes in vitro. Genes identi¢ed include cytokines and in£ammatory mediators, extracellular matrix and adhesion molecules, cell cycle and proliferation related proteins, transcription related proteins, and apoptotic mediators. OM identi-¢ed 1185 di¡erentially expressed genes in TNF-K K-treated synoviocytes. The regulation of Nef-associated factor-1 (Naf1), an A20-binding, nuclear factor kappa B (NFU UB) inhibitory protein was probed further given its putative role as an endogenous brake for the expression of some TNF-K K-driven genes. Naf1 mRNA levels were higher in synovial biopsies from patients with active RA and seronegative arthropathy than in those from patients with osteoarthritis. These ¢ndings underscore the value of transcriptome analysis in cytokine-activated synoviocyte cultures in vitro as a means of identifying disease-associated genes in human arthritis, and implicate Naf1 as a potential modulator of TNF-K K bioactivity in RA. ß
Tumor cell migration plays a central role in the process of cancer metastasis. We recently identi... more Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 receptor or Galpha(s) expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr-34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Galpha(s)/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.
Journal of mammary gland biology and neoplasia, 2006
During puberty the mammary gland develops from a rudimentary tree to a branched epithelial networ... more During puberty the mammary gland develops from a rudimentary tree to a branched epithelial network of ducts which can support alveolar development and subsequent milk production during pregnancy and lactation. This process involves growth, proliferation, migration, branching, invasion, apoptosis and above all, tight regulation which allows these processes to take place simultaneously during the course of just a few weeks to create an adult gland. The process is under hormonal control and is thus coordinated with reproductive development. Mouse models, with overexpressed or knocked-out genes, have highlighted a number of pubertal mammary gland phenotypes and given significant insight into the regulatory mechanisms controlling this period of development. Here we review the published findings of the wide range of gene-manipulated mammary mouse models, documenting the common pubertal mammary gland phenotypes observed, and summarizing their contribution to our current understanding of how pubertal mammary gland development occurs.
Journal of the American Society of Nephrology : JASN, 2008
Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by h... more Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.
Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In... more Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells -but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation. In the present study we sought to investigate whether negative targeting of TNK2 by siRNA could be used to inhibit cancer cell invasion, to establish the contribution of its effect on the EGFR and to consequently attempt to resolve the issue of TNK2's mechanism of action.
Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased drama... more Utilisation of ‘omics’ technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets. (Part of a Multi-author Review)
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse ma... more Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-b and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERa binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERa localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERa. Similar to ERa, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERa-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Abstract The influential role of Wnt5a in tumor progression underscores the requirement for devel... more Abstract The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63.
Abstract. Utilisation of omics technologies, in particular gene expression profiling, has increas... more Abstract. Utilisation of omics technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, highthroughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers.
Abstract Expression microarray analysis identified over 930 genes regulated during puberty in the... more Abstract Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented.
Abstract Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prog... more Abstract Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells–but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation.
Abstract Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregul... more Abstract Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects.
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Papers by Jill Howlin