Under normal conditions, the most significant expansion and differentiation of the adult mammary ... more Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into...
It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenv... more It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the dete...
The transcription factor signal transducer and activator of transcription 3 (STAT3) is constituti... more The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knockin mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumorderived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558-67.
The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately trans... more The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings re...
The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While S... more The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While STAT3 promotes cell survival/proliferation, motility and immune tolerance and is considered as an oncogene, STAT1 mostly triggers anti-proliferative and pro-apoptotic responses while enhancing anti-tumor immunity. Despite being activated downstream of common cytokine and growth factor receptors, their activation is reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1 and STAT3 activation in tumorigenesis and their potential cross-regulation mechanisms.
Downstream of cytokine or growth factor receptors, STAT3 counteracts inflammation and promotes ce... more Downstream of cytokine or growth factor receptors, STAT3 counteracts inflammation and promotes cell survival/proliferation and immune tolerance while STAT1 inhibits proliferation and favours innate and adaptive immune responses. STAT1 and STAT3 activation are reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1/3 activation and discuss the hypothesis that perturbation of their expression and/or activation levels may provide novel therapeutic strategies in different clinical settings and particularly in cancer.
Journal of Mammary Gland Biology and Neoplasia, 2009
The transcription factor Stat3 is essential for timely initiation of post-lactational regression ... more The transcription factor Stat3 is essential for timely initiation of post-lactational regression and orchestrates the processes of cell death and tissue remodelling that occur during the first 6 days of involution in the mouse. Paradoxically, STAT3 is also frequently found to be constitutively active in breast cancer and tumors can become addicted to STAT3. This raises two interesting questions: 1) do the high levels of active Stat3 present in the mammary epithelium during involution promote tumor spread and 2) how do tumor cells escape the pro-apoptotic effects of Stat3? In order to address these questions, it is essential to understand the role of Stat3 in involution and the mechanisms by which Stat3 regulates both cell death and tissue remodelling. A number of studies have been undertaken using genetically modified mice and microarray analyses and two significant findings arose from these investigations. Firstly, post-lactational regression is associated with an acute phase and inflammatory response in addition to cell death and secondly, Stat3 alone is insufficient to induce involution in the absence of the NF-kappaB regulatory kinase IKKbeta. Both Stat3 and NF-kappaB have been shown to regulate the expression of genes involved in inflammatory signalling and the acute phase response. These findings suggest a role for the innate immune response in mammary epithelial cell fate during involution and highlight potential roles for this response in tissue remodelling-associated breast cancer metastasis.
Hormone Molecular Biology and Clinical Investigation, 2000
The pro-oncogenic transcription factor STAT3 is constitutively active in tumours of many differen... more The pro-oncogenic transcription factor STAT3 is constitutively active in tumours of many different origins, which often become addicted to its activity. STAT3 is believed to contribute to the initial survival of pre-cancerous cells as well as to hyper-proliferation and, later, metastasis. Materials and methods: To evaluate the contribution of enhanced STAT3 activation in a controlled model system, we generated knock-in mice in which a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele and analysed the contribution of the allele to breast tumorigenesis. Moreover, we generated Stat3 C/C MEF cells and analy sed their gene expression and metabolic profi les. Results: Constitutively active STAT3 could enhance the tumorigenic power of the rat Neu oncogene in MMTV-Neu transgenic mice and trigger the production of earlier onset and more invasive mammary tumours. Tumour-derived cell lines displayed higher migrating, invading and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by STAT3-dependent over-expression of the C-terminal tensin-like (Cten) focal adhesion protein. Moreover, STAT3C alone was able to induce aerobic glycolysis and down-regulate mitochondrial activity, both in primary fi broblasts and in STAT3-dependent tumour cell lines, acting via both HIF-1 α -dependent and independent mechanisms. Conclusions: STAT3 can induce a metabolic switch that predisposes cells to aberrant survival, enhanced proliferation and, fi nally, tumour transformation. Later, enhanced Cten Q1: Genes are set in italic and proteins are set in roman. Please ensure that italic/ roman have been applied correctly throughout. Q2: Please provide manufacturer's name, city, state, country for all reagents, devices, and software mentioned throughout.
Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat)... more Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development, and this pathway is frequently hyper-activated in cancer, including tumours of the breast. Stats 3, 5 and 6 have important roles in the differentiation and survival of mammary alveolar cells, but somewhat paradoxically, both Stat3 and 5 can have oncogenic activity in the mammary gland. Constitutive activation of JAK2 could be anticipated to result in hyper-activation of Stats 1, 3, 5 and 6 with concomitant cell transformation, although the outcome is difficult to envisage, particularly since Stats 3 and 5 play opposing roles in normal mammary gland development. Here, we show that expression of a constitutively active JAK2 mutant, JAK2 V617F, leads to hyper-activation of Stat5 in mammary epithelial cells (MECs), and transgenic mice expressing JAK2 V617F specifically in the mammary gland exhibit accelerated alveologenesis during pregnancy and delayed post-lactational regression. Overexpressing JAK2 V617F in MECs in vitro results in elevated proliferation and resistance to cell death. Furthermore, constitutively active JAK2 enhances anchorage-independent cell growth in the presence of a co-operating oncogene and accelerates tumourigenesis in a xenograft model. Taken together, our results provide insights into signalling downstream of constitutively active JAK2 and could be important for understanding the molecular mechanisms of breast tumourigenesis. (MMC) These authors contributed equally to this work.
The transcription factor signal transducer and activator of transcription 3 (STAT3) is constituti... more The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knockin mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumorderived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558-67.
Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during po... more Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors. Citation: Staniszewska AD, Pensa S, Caffarel MM, Anderson LH, Poli V, et al. (2012) Stat3 Is Required to Maintain the Full Differentiation Potential of Mammary Stem Cells and the Proliferative Potential of Mammary Luminal Progenitors. PLoS ONE 7(12): e52608.
Under normal conditions, the most significant expansion and differentiation of the adult mammary ... more Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into...
It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenv... more It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the dete...
The transcription factor signal transducer and activator of transcription 3 (STAT3) is constituti... more The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knockin mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumorderived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558-67.
The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately trans... more The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings re...
The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While S... more The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While STAT3 promotes cell survival/proliferation, motility and immune tolerance and is considered as an oncogene, STAT1 mostly triggers anti-proliferative and pro-apoptotic responses while enhancing anti-tumor immunity. Despite being activated downstream of common cytokine and growth factor receptors, their activation is reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1 and STAT3 activation in tumorigenesis and their potential cross-regulation mechanisms.
Downstream of cytokine or growth factor receptors, STAT3 counteracts inflammation and promotes ce... more Downstream of cytokine or growth factor receptors, STAT3 counteracts inflammation and promotes cell survival/proliferation and immune tolerance while STAT1 inhibits proliferation and favours innate and adaptive immune responses. STAT1 and STAT3 activation are reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1/3 activation and discuss the hypothesis that perturbation of their expression and/or activation levels may provide novel therapeutic strategies in different clinical settings and particularly in cancer.
Journal of Mammary Gland Biology and Neoplasia, 2009
The transcription factor Stat3 is essential for timely initiation of post-lactational regression ... more The transcription factor Stat3 is essential for timely initiation of post-lactational regression and orchestrates the processes of cell death and tissue remodelling that occur during the first 6 days of involution in the mouse. Paradoxically, STAT3 is also frequently found to be constitutively active in breast cancer and tumors can become addicted to STAT3. This raises two interesting questions: 1) do the high levels of active Stat3 present in the mammary epithelium during involution promote tumor spread and 2) how do tumor cells escape the pro-apoptotic effects of Stat3? In order to address these questions, it is essential to understand the role of Stat3 in involution and the mechanisms by which Stat3 regulates both cell death and tissue remodelling. A number of studies have been undertaken using genetically modified mice and microarray analyses and two significant findings arose from these investigations. Firstly, post-lactational regression is associated with an acute phase and inflammatory response in addition to cell death and secondly, Stat3 alone is insufficient to induce involution in the absence of the NF-kappaB regulatory kinase IKKbeta. Both Stat3 and NF-kappaB have been shown to regulate the expression of genes involved in inflammatory signalling and the acute phase response. These findings suggest a role for the innate immune response in mammary epithelial cell fate during involution and highlight potential roles for this response in tissue remodelling-associated breast cancer metastasis.
Hormone Molecular Biology and Clinical Investigation, 2000
The pro-oncogenic transcription factor STAT3 is constitutively active in tumours of many differen... more The pro-oncogenic transcription factor STAT3 is constitutively active in tumours of many different origins, which often become addicted to its activity. STAT3 is believed to contribute to the initial survival of pre-cancerous cells as well as to hyper-proliferation and, later, metastasis. Materials and methods: To evaluate the contribution of enhanced STAT3 activation in a controlled model system, we generated knock-in mice in which a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele and analysed the contribution of the allele to breast tumorigenesis. Moreover, we generated Stat3 C/C MEF cells and analy sed their gene expression and metabolic profi les. Results: Constitutively active STAT3 could enhance the tumorigenic power of the rat Neu oncogene in MMTV-Neu transgenic mice and trigger the production of earlier onset and more invasive mammary tumours. Tumour-derived cell lines displayed higher migrating, invading and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by STAT3-dependent over-expression of the C-terminal tensin-like (Cten) focal adhesion protein. Moreover, STAT3C alone was able to induce aerobic glycolysis and down-regulate mitochondrial activity, both in primary fi broblasts and in STAT3-dependent tumour cell lines, acting via both HIF-1 α -dependent and independent mechanisms. Conclusions: STAT3 can induce a metabolic switch that predisposes cells to aberrant survival, enhanced proliferation and, fi nally, tumour transformation. Later, enhanced Cten Q1: Genes are set in italic and proteins are set in roman. Please ensure that italic/ roman have been applied correctly throughout. Q2: Please provide manufacturer's name, city, state, country for all reagents, devices, and software mentioned throughout.
Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat)... more Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development, and this pathway is frequently hyper-activated in cancer, including tumours of the breast. Stats 3, 5 and 6 have important roles in the differentiation and survival of mammary alveolar cells, but somewhat paradoxically, both Stat3 and 5 can have oncogenic activity in the mammary gland. Constitutive activation of JAK2 could be anticipated to result in hyper-activation of Stats 1, 3, 5 and 6 with concomitant cell transformation, although the outcome is difficult to envisage, particularly since Stats 3 and 5 play opposing roles in normal mammary gland development. Here, we show that expression of a constitutively active JAK2 mutant, JAK2 V617F, leads to hyper-activation of Stat5 in mammary epithelial cells (MECs), and transgenic mice expressing JAK2 V617F specifically in the mammary gland exhibit accelerated alveologenesis during pregnancy and delayed post-lactational regression. Overexpressing JAK2 V617F in MECs in vitro results in elevated proliferation and resistance to cell death. Furthermore, constitutively active JAK2 enhances anchorage-independent cell growth in the presence of a co-operating oncogene and accelerates tumourigenesis in a xenograft model. Taken together, our results provide insights into signalling downstream of constitutively active JAK2 and could be important for understanding the molecular mechanisms of breast tumourigenesis. (MMC) These authors contributed equally to this work.
The transcription factor signal transducer and activator of transcription 3 (STAT3) is constituti... more The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knockin mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)-Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumorderived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558-67.
Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during po... more Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors. Citation: Staniszewska AD, Pensa S, Caffarel MM, Anderson LH, Poli V, et al. (2012) Stat3 Is Required to Maintain the Full Differentiation Potential of Mammary Stem Cells and the Proliferative Potential of Mammary Luminal Progenitors. PLoS ONE 7(12): e52608.
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Papers by S. Pensa