Hidetoshi Mori - Academia.edu (original) (raw)

Papers by Hidetoshi Mori

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development th... more Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout of Mmp14 (KO) in mice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organmodel for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into an unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO...

Frontiers in Cell and Developmental Biology, 2018

Breast Cancer Research, 2017

Background: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen recep... more Background: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER +), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 −/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. Methods: Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129: Stat1 −/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. Results: Tumorigenesis in 129:Stat1 −/− originates from a population of FoxA1 + large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 −/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. Conclusions: 129:Stat1 −/− is a unique model for studying the critical origins and risk reduction strategies in agerelated ER + breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Frontiers in Cell and Developmental Biology, 2016

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma re... more Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pH i) measured using the fluorescent ratio dye 2 ′ ,7 ′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pH i with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pH i that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pH i similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pH i similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional Lobo et al. Metabolic Heterogeneity in Mouse Mammary Precancer microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Proceedings of the National Academy of Sciences, 2016

Significance Malignant cells of breast carcinoma and nonmalignant epithelia of branching mammary ... more Significance Malignant cells of breast carcinoma and nonmalignant epithelia of branching mammary glands share the ability to migrate through their surroundings. To form the mammary tree-like architecture, nonmalignant epithelia must migrate in a controlled fashion, integrating cues from their microenvironment, notably, the glycan appendages on extracellular proteins and lipids. Here, we show that Galectin-1, a glycan-binding protein, is able to sense glycan signatures on mammary gland epithelia, transmit this information to epithelial nuclei by direct translocation, and drive branching migration. Nuclear galectin-1 is regulated by the relative levels of α2,6–sialic acids and N -acetyllactosamine on extracellular glycans. Similar lectin–glycan signatures were observed in malignant breast cells and suggest cancer cells use this pathway during their invasion.

Biochemical Society Symposia, 2003

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that perfor... more Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that performs processing of cell surface proteins and degradation of extracellular matrix (ECM) components. Through these proteolytic events, MT1-MMP regulates various cellular functions, including ECM turnover, promotion of cell migration and invasion, and morphogenic responses to extracellular stimuli. MT1-MMP has to be regulated strictly to accomplish its function appropriately at various steps, including at the transcriptional and post-translational levels. MT1-MMP was originally identified as an invasion-promoting enzyme expressed in malignant tumour cells, and also as a specific activator of proMMP-2, which is believed to play a role in invasion of the basement membrane. Since then, it has attracted attention as a membrane-associated MMP that promotes cancer cell invasion and angiogenesis by endothelial cells. Although MT1-MMP has now become one of the best characterized enzymes in the MMP fa...

Oncogene, 2004

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environm... more Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environment through its proteolytic activity and promotes migration, invasion, and proliferation of tumor cells. During cell migration, MT1-MMP binds to CD44H, a major hyaluronan receptor, through the hemopexin-like (HPX) domain and localizes at the migration front. MT1-MMP is also responsible for shedding CD44H, which supports CD44H-mediated cell migration. In this study, we asked whether the binding of MT1-MMP to CD44H is a prerequisite step for the successive shedding. Deletion of the HPX domain deprived MT1-MMP of its shedding activity. Furthermore, disruption of the CD44H/MT1-MMP complex by overexpressing the HPX fragments resulted in inhibition of the shedding. Thus, the CD44H in the complex appears to be the direct substrate of MT1-MMP for shedding. Interestingly, other members of the MT-MMP family showed varied extents of CD44H shedding. Domain swapping between MT1-MMP and other MT-MMPs revealed that the ability of the HPX domains to bind CD44H is conserved among them. However, the shedding activity was different depending on the catalytic domains. The conserved binding ability of the HPX domains suggests that CD44H may act as a core molecule assembling multiple MT-MMPs on the cell surface.

Journal of Biological Chemistry, 1999

Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs ... more Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs have about a 20-amino acid cytoplasmic tail following the transmembrane domain. In contrast, a putative transmembrane domain of MT4-MMP locates at the very C-terminal end, and the expected cytoplasmic tail is very short or nonexistent. Such sequences often act as a glycosylphosphatidylinositol (GPI) anchoring signal rather than as a transmembrane domain. We thus examined the possibility that MT4-MMP is a GPI-anchored proteinase. Our results showed that [ 3 H]ethanolamine, which can be incorporated into the GPI unit, specifically labeled the MT4-MMP C-terminal end in a sequencedependent manner. In addition, phosphatidylinositolspecific phospholipase C treatment released the MT4-MMP from the surface of transfected cells. These results indicate that MT4-MMP is the first GPI-anchored proteinase in the MMP family. During cultivation of the transfected cells, MT4-MMP appeared to be shed from the cell surface by the action of an endogenous metalloproteinase. GPI anchoring of MT4-MMP on the cell surface indicates a unique biological function and character for this proteinase.

PeerJ, 2016

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development th... more Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout ofMmp14(KO)inmice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organ model for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into an unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO ru...

SummaryDuctal Carcinoma in situ (DCIS) is an avascular disease characterized by profound acidosis... more SummaryDuctal Carcinoma in situ (DCIS) is an avascular disease characterized by profound acidosis. Pre-malignant cells within this niche must adapt to acidosis to survive and thrive. A component of this acid-adaptation involves extracellular matrix remodeling leading to niche construction and remodeling. Using discovery proteomics, we identified that collagen producing enzyme PLODs are upregulated in acid-adapted breast cancer cells. Second harmonic generation microscopy showed significant collagen deposition within DCIS lesions of patients. In vitro analyses identified that acid-adaptation involves production of rare collagens that can be regulated by Ras and SMAD pathway. Secretome analysis showed upregulation ECM remodeling enzymes such as TGM2 and LOXL2. Comparison of acid induced collagens in vitro and in patient data showed correlation between rare collagens production and survival of patients. We conclude acidosis induces collagen production by cancer cells and promote growth...

Toxicologic pathology, Jan 7, 2015

Tissue localization of immune cells is critical to the study of disease processes in mouse models... more Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can ...

Tissue organogenesis is directed by both intercellular interactions and communication with the su... more Tissue organogenesis is directed by both intercellular interactions and communication with the surrounding microenvironment. When cells are cultured on twodimensional plastic substrata (2D), important signals controlling programs of cell proliferation, metabolism, differentiation and death responsible for the formation of correct tissue-specific architecture and function are lost. Designing three-dimensional (3D), physiologically relevant culture models, we can recapitulate some crucial aspects of the dynamic and reciprocal signaling necessary for establishing and maintaining tissue specific morphogenic programs. Here we briefly describe the details of robust methods for culturing mouse primary mammary organoids in 3D gels of different extracellular matrices and describe techniques for analyzing the resulting structures. These designer microenvironments are useful for both understanding branching morphogenesis and signaling integrations, but also for analysis of individual susceptibilities and drug testing.

For decades, the work of cell and developmental biologists has demonstrated the striking ability ... more For decades, the work of cell and developmental biologists has demonstrated the striking ability of the mesenchyme and the stroma to instruct epithelial form and function in the mammary gland, but the role of extracellular matrix (ECM) molecules in mammary pattern specification has not been elucidated. Here, we show that stromal collagen I (Col-I) fibers in the mammary fat pad are axially oriented prior to branching morphogenesis. Upon puberty, the branching epithelium orients along these fibers, thereby adopting a similar axial bias. To establish a causal relationship from Col-I fiber to epithelial orientation, we embedded mammary organoids within axially oriented Col-I fiber gels and observed dramatic epithelial co-orientation. Whereas a constitutively active form of Rac1, a molecule implicated in cell motility, prevented a directional epithelial response to Col-I fiber orientation, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway did not. However, time-lapse studies revealed that, within randomly oriented Col-I matrices, the epithelium axially aligns fibers at branch sites via RhoA/ROCK-mediated contractions. Our data provide an explanation for how the stromal ECM encodes architectural cues for branch orientation as well as how the branching epithelium interprets and reinforces these cues through distinct signaling processes.

Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intrace... more Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy.

Developmental Biology, 2007

Transforming growth factor-α (TGFα) and fibroblast growth factor-7 (FGF7) exhibit distinct expres... more Transforming growth factor-α (TGFα) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPKERK1,2); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPKERK1,2 for 1 h, induced by TGFα, was necessary and

Integrative Biology, 2011

Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is cru... more Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is crucial to the initial stages of branching morphogenesis, and a hallmark of tissue repair and dissemination of certain tumors. The collagenous ECM acts as a mechanical barrier against CCI. However, the physical nature of this barrier and how it is overcome by cells remains incompletely understood. To address these questions, we performed theoretical and experimental analysis of mammary epithelial branching morphogenesis in 3D type I collagen (collagen-I) gels. We found that the mechanical resistance of collagen-I is largely due to its elastic rather than its viscous properties. We also identified two strategies utilized by mammary epithelial cells that can independently minimize ECM mechanical resistance during CCI. First, cells adopt a narrow tube-like geometry during invasion, which minimizes the elastic opposition from the ECM as revealed by theoretical modeling of the most frequent invasive shapes and sizes. Second, the stiffness of the collagenous ECM is reduced at invasive fronts due to its degradation by matrix metalloproteinases (MMPs), as indicated by direct measurements of collagen-I microelasticity by atomic force microscopy. Molecular techniques further specified that the membrane-bound MMP14 mediates degradation of collagen-I at invasive fronts. Thus, our findings reveal that MMP14 is necessary to efficiently reduce the physical restraints imposed by collagen-I during branching morphogenesis, and help our overall understanding of how forces are balanced between cells and their surrounding ECM to maintain collective geometry and mechanical stability during CCI.

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development th... more Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout of Mmp14 (KO) in mice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organmodel for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into an unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO...

Frontiers in Cell and Developmental Biology, 2018

Breast Cancer Research, 2017

Background: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen recep... more Background: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER +), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 −/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. Methods: Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129: Stat1 −/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. Results: Tumorigenesis in 129:Stat1 −/− originates from a population of FoxA1 + large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 −/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. Conclusions: 129:Stat1 −/− is a unique model for studying the critical origins and risk reduction strategies in agerelated ER + breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Frontiers in Cell and Developmental Biology, 2016

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma re... more Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pH i) measured using the fluorescent ratio dye 2 ′ ,7 ′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pH i with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pH i that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pH i similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pH i similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional Lobo et al. Metabolic Heterogeneity in Mouse Mammary Precancer microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Proceedings of the National Academy of Sciences, 2016

Significance Malignant cells of breast carcinoma and nonmalignant epithelia of branching mammary ... more Significance Malignant cells of breast carcinoma and nonmalignant epithelia of branching mammary glands share the ability to migrate through their surroundings. To form the mammary tree-like architecture, nonmalignant epithelia must migrate in a controlled fashion, integrating cues from their microenvironment, notably, the glycan appendages on extracellular proteins and lipids. Here, we show that Galectin-1, a glycan-binding protein, is able to sense glycan signatures on mammary gland epithelia, transmit this information to epithelial nuclei by direct translocation, and drive branching migration. Nuclear galectin-1 is regulated by the relative levels of α2,6–sialic acids and N -acetyllactosamine on extracellular glycans. Similar lectin–glycan signatures were observed in malignant breast cells and suggest cancer cells use this pathway during their invasion.

Biochemical Society Symposia, 2003

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that perfor... more Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that performs processing of cell surface proteins and degradation of extracellular matrix (ECM) components. Through these proteolytic events, MT1-MMP regulates various cellular functions, including ECM turnover, promotion of cell migration and invasion, and morphogenic responses to extracellular stimuli. MT1-MMP has to be regulated strictly to accomplish its function appropriately at various steps, including at the transcriptional and post-translational levels. MT1-MMP was originally identified as an invasion-promoting enzyme expressed in malignant tumour cells, and also as a specific activator of proMMP-2, which is believed to play a role in invasion of the basement membrane. Since then, it has attracted attention as a membrane-associated MMP that promotes cancer cell invasion and angiogenesis by endothelial cells. Although MT1-MMP has now become one of the best characterized enzymes in the MMP fa...

Oncogene, 2004

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environm... more Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environment through its proteolytic activity and promotes migration, invasion, and proliferation of tumor cells. During cell migration, MT1-MMP binds to CD44H, a major hyaluronan receptor, through the hemopexin-like (HPX) domain and localizes at the migration front. MT1-MMP is also responsible for shedding CD44H, which supports CD44H-mediated cell migration. In this study, we asked whether the binding of MT1-MMP to CD44H is a prerequisite step for the successive shedding. Deletion of the HPX domain deprived MT1-MMP of its shedding activity. Furthermore, disruption of the CD44H/MT1-MMP complex by overexpressing the HPX fragments resulted in inhibition of the shedding. Thus, the CD44H in the complex appears to be the direct substrate of MT1-MMP for shedding. Interestingly, other members of the MT-MMP family showed varied extents of CD44H shedding. Domain swapping between MT1-MMP and other MT-MMPs revealed that the ability of the HPX domains to bind CD44H is conserved among them. However, the shedding activity was different depending on the catalytic domains. The conserved binding ability of the HPX domains suggests that CD44H may act as a core molecule assembling multiple MT-MMPs on the cell surface.

Journal of Biological Chemistry, 1999

Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs ... more Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs have about a 20-amino acid cytoplasmic tail following the transmembrane domain. In contrast, a putative transmembrane domain of MT4-MMP locates at the very C-terminal end, and the expected cytoplasmic tail is very short or nonexistent. Such sequences often act as a glycosylphosphatidylinositol (GPI) anchoring signal rather than as a transmembrane domain. We thus examined the possibility that MT4-MMP is a GPI-anchored proteinase. Our results showed that [ 3 H]ethanolamine, which can be incorporated into the GPI unit, specifically labeled the MT4-MMP C-terminal end in a sequencedependent manner. In addition, phosphatidylinositolspecific phospholipase C treatment released the MT4-MMP from the surface of transfected cells. These results indicate that MT4-MMP is the first GPI-anchored proteinase in the MMP family. During cultivation of the transfected cells, MT4-MMP appeared to be shed from the cell surface by the action of an endogenous metalloproteinase. GPI anchoring of MT4-MMP on the cell surface indicates a unique biological function and character for this proteinase.

PeerJ, 2016

Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development th... more Membrane-anchored matrix metalloproteinase 14 (MMP14) is involved broadly in organ development through both its proteolytic and signal-transducing functions. Knockout ofMmp14(KO)inmice results in a dramatic reduction of body size and wasting followed by premature death, the mechanism of which is poorly understood. Since the mammary gland develops after birth and is thus dependent for its functional progression on systemic and local cues, we chose it as an organ model for understanding why KO mice fail to thrive. A global analysis of the mammary glands’ proteome in the wild type (WT) and KO mice provided insight into an unexpected role of MMP14 in maintaining metabolism and homeostasis. We performed mass spectrometry and quantitative proteomics to determine the protein signatures of mammary glands from 7 to 11 days old WT and KO mice and found that KO rudiments had a significantly higher level of rate-limiting enzymes involved in catabolic pathways. Glycogen and lipid levels in KO ru...

SummaryDuctal Carcinoma in situ (DCIS) is an avascular disease characterized by profound acidosis... more SummaryDuctal Carcinoma in situ (DCIS) is an avascular disease characterized by profound acidosis. Pre-malignant cells within this niche must adapt to acidosis to survive and thrive. A component of this acid-adaptation involves extracellular matrix remodeling leading to niche construction and remodeling. Using discovery proteomics, we identified that collagen producing enzyme PLODs are upregulated in acid-adapted breast cancer cells. Second harmonic generation microscopy showed significant collagen deposition within DCIS lesions of patients. In vitro analyses identified that acid-adaptation involves production of rare collagens that can be regulated by Ras and SMAD pathway. Secretome analysis showed upregulation ECM remodeling enzymes such as TGM2 and LOXL2. Comparison of acid induced collagens in vitro and in patient data showed correlation between rare collagens production and survival of patients. We conclude acidosis induces collagen production by cancer cells and promote growth...

Toxicologic pathology, Jan 7, 2015

Tissue localization of immune cells is critical to the study of disease processes in mouse models... more Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can ...

Tissue organogenesis is directed by both intercellular interactions and communication with the su... more Tissue organogenesis is directed by both intercellular interactions and communication with the surrounding microenvironment. When cells are cultured on twodimensional plastic substrata (2D), important signals controlling programs of cell proliferation, metabolism, differentiation and death responsible for the formation of correct tissue-specific architecture and function are lost. Designing three-dimensional (3D), physiologically relevant culture models, we can recapitulate some crucial aspects of the dynamic and reciprocal signaling necessary for establishing and maintaining tissue specific morphogenic programs. Here we briefly describe the details of robust methods for culturing mouse primary mammary organoids in 3D gels of different extracellular matrices and describe techniques for analyzing the resulting structures. These designer microenvironments are useful for both understanding branching morphogenesis and signaling integrations, but also for analysis of individual susceptibilities and drug testing.

For decades, the work of cell and developmental biologists has demonstrated the striking ability ... more For decades, the work of cell and developmental biologists has demonstrated the striking ability of the mesenchyme and the stroma to instruct epithelial form and function in the mammary gland, but the role of extracellular matrix (ECM) molecules in mammary pattern specification has not been elucidated. Here, we show that stromal collagen I (Col-I) fibers in the mammary fat pad are axially oriented prior to branching morphogenesis. Upon puberty, the branching epithelium orients along these fibers, thereby adopting a similar axial bias. To establish a causal relationship from Col-I fiber to epithelial orientation, we embedded mammary organoids within axially oriented Col-I fiber gels and observed dramatic epithelial co-orientation. Whereas a constitutively active form of Rac1, a molecule implicated in cell motility, prevented a directional epithelial response to Col-I fiber orientation, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway did not. However, time-lapse studies revealed that, within randomly oriented Col-I matrices, the epithelium axially aligns fibers at branch sites via RhoA/ROCK-mediated contractions. Our data provide an explanation for how the stromal ECM encodes architectural cues for branch orientation as well as how the branching epithelium interprets and reinforces these cues through distinct signaling processes.

Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intrace... more Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy.

Developmental Biology, 2007

Transforming growth factor-α (TGFα) and fibroblast growth factor-7 (FGF7) exhibit distinct expres... more Transforming growth factor-α (TGFα) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPKERK1,2); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPKERK1,2 for 1 h, induced by TGFα, was necessary and

Integrative Biology, 2011

Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is cru... more Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is crucial to the initial stages of branching morphogenesis, and a hallmark of tissue repair and dissemination of certain tumors. The collagenous ECM acts as a mechanical barrier against CCI. However, the physical nature of this barrier and how it is overcome by cells remains incompletely understood. To address these questions, we performed theoretical and experimental analysis of mammary epithelial branching morphogenesis in 3D type I collagen (collagen-I) gels. We found that the mechanical resistance of collagen-I is largely due to its elastic rather than its viscous properties. We also identified two strategies utilized by mammary epithelial cells that can independently minimize ECM mechanical resistance during CCI. First, cells adopt a narrow tube-like geometry during invasion, which minimizes the elastic opposition from the ECM as revealed by theoretical modeling of the most frequent invasive shapes and sizes. Second, the stiffness of the collagenous ECM is reduced at invasive fronts due to its degradation by matrix metalloproteinases (MMPs), as indicated by direct measurements of collagen-I microelasticity by atomic force microscopy. Molecular techniques further specified that the membrane-bound MMP14 mediates degradation of collagen-I at invasive fronts. Thus, our findings reveal that MMP14 is necessary to efficiently reduce the physical restraints imposed by collagen-I during branching morphogenesis, and help our overall understanding of how forces are balanced between cells and their surrounding ECM to maintain collective geometry and mechanical stability during CCI.