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Kelly M. Mcnagny - One of the best experts on this subject based on the ideXlab platform.
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abstract 4911 characterizing the role of Podocalyxin s cytoplasmic tail domain in collective tumor invasion
Cancer Research, 2020Co-Authors: Erin M Bell, Kelly M. Mcnagny, Marcia L Graves, Pamela Dean, Calvin D RoskelleyAbstract:High expression of the single-pass transmembrane sialomucin, Podocalyxin, has been shown by many groups to correlate with poor disease outcome in a number of solid tumor types, including colorectal, ovarian, pancreatic and breast cancers. We had previously identified that high Podocalyxin expression in invasive ductal breast carcinoma was an independent indicator of poor survival (Somasiri et al., 2004, Cancer Res. 64:15). Further analysis of these tumor samples revealed that this decrease in survival occurred without the loss of membranous, junctional E-cadherin, suggesting that these tumors may have invaded collectively without necessitating an overt epithelial to mesenchymal transition (EMT). Experimentally, forced over-expression of Podocalyxin in polarity-disrupted human MCF7 breast cancer cells drives the formation of invasive orthotopic xenograft tumors and elongated, cohesive, and E-Cadherin-expressing spheroids in three-dimensional (3D) culture as compared to control (Graves et al., 2016, Breast Canc. Res. 18:11). Further, treatment of these Podocalyxin-overexpressing MCF7 cells with the myosin inhibitor, blebbistatin, and the small molecule inhibitor of ezrin-actin binding, NSC668394, resulted in decreased collective invasion and migration, respectively. Together these data suggest that Podocalyxin, through interactions with the actin cytoskeleton via its cytoplasmic tail binding partners, can facilitate increased collective epithelial tumor cell motility, at least in some contexts. To test this hypothesis, we generated Podocalyxin null MCF7 clones and cell populations using CRISPR-Cas9 genome editing and reconstituted these cells with mutant forms of Podocalyxin that are unable to interact with the scaffolding proteins NHERF and/or ezrin and hence with the actin cytoskeleton. Preliminary results from 3D culture and live imaging of these mutant Podocalyxin-expressing cells suggests that loss of Podocalyxin9s cytoplasmic tail results in decreased spheroid invasion that may be a result of deficiencies in actomyosin contractility. Hence, increased expression and mislocalization of Podocalyxin may facilitate aberrant interactions with the actin cytoskeleton and contractile machinery, driving enhanced cell motility and, in certain tumor microenvironments, promote collective tumor invasion. Citation Format: Erin M. Bell, Marcia L. Graves, Pamela Dean, Kelly M. McNagny, Calvin D. Roskelley. Characterizing the role of Podocalyxin9s cytoplasmic tail domain in collective tumor invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4911.
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distinct functional requirements for Podocalyxin in immature and mature podocytes reveal mechanisms of human kidney disease
Scientific Reports, 2020Co-Authors: Ido Refaeli, Michael R Hughes, Calvin D Roskelley, Wayne A Vogl, Alvin Kawai Wong, Mei Lin Z Bissonnette, Sean J Barbour, Benjamin S Freedman, Kelly M. McnagnyAbstract:Dominant and recessive mutations in Podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and Podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for Podocalyxin in all tissues (Podxl+/-). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl-/-), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/- mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/- mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which Podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/- mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.
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the first identified heterozygous nonsense mutations in Podocalyxin offer new perspectives on the biology of podocytopathies
Clinical Science, 2019Co-Authors: Ido Refaeli, Michael R Hughes, Kelly M. McnagnyAbstract:In the last two decades, our understanding of the genetic underpinnings of inherited podocytopathies has advanced immensely. By sequencing the genomes of a large pool of families affected by focal segmental glomerulosclerosis (FSGS), researchers have identified a common theme: familial podocytopathies are frequently caused by genes selectively expressed in podocytes. Podocalyxin is a podocyte-specific surface sialomucin that has long been known to play important roles in podocyte morphogenesis and function. Few studies, however, have shown a conclusive link between mutations in the gene and FSGS complemented by functional evidence. In a fascinating new paper published in Clinical Science, Lin et al. identify two unrelated pedigrees in which dominant loss-of-function mutations in PODXL lead to adult-onset FSGS. Nonsense-mediated decay of the mutated PODXL transcripts leads to protein insufficiency, which in turn cause podocyte dysfunction through defects in motility and cytoskeletal organization. This is the first study to date that demonstrates, mechanistically, how autosomal dominant mutations in Podocalyxin can lead to FSGS and renal insufficiency. Here, we summarize the experimental findings of this manuscript and propose, perhaps, a more controversial hypothesis: down-regulation of Podocalyxin protein expression from podocytes is a critical turning point in the progression of most podocytopathies and may be mechanistically relevant to glomerulopathies in which podocyte damage is not necessarily induced by genetic lesions.
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loss of Podocalyxin causes a novel syndromic type of congenital nephrotic syndrome
Experimental and Molecular Medicine, 2017Co-Authors: Kelly M. Mcnagny, Michael R Hughes, Hee Gyung Kang, Moses Lee, Kyoung Boon Lee, Bo Sang Kwon, Sangmoon Lee, Yo Han AhnAbstract:Many cellular structures directly imply specific biological functions. For example, normal slit diaphragm structures that extend from podocyte foot processes ensure the filtering function of renal glomeruli. These slits are covered by a number of surface proteins, such as nephrin, podocin, Podocalyxin and CD2AP. Here we report a human patient presenting with congenital nephrotic syndrome, omphalocele and microcoria due to two loss-of-function mutations in PODXL, which encodes Podocalyxin, inherited from each parent. This set of symptoms strikingly mimics previously reported mouse Podxl-/- embryos, emphasizing the essential function of PODXL in mammalian kidney development and highlighting this patient as a human PODXL-null model. The results underscore the utility of current genomics approaches to provide insights into the genetic mechanisms of human disease traits through molecular diagnosis.
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RESEARCH ARTICLE Open Access The cell surface mucin Podocalyxin regulates collective breast tumor budding
2016Co-Authors: Marcia L Graves, Kelly M. Mcnagny, Julie S Nielsen, Blake C Gilks, Pamela Austin, Erin M Bell, Jane A. Cipollone, Calvin D RoskelleyAbstract:Background: Overexpression of the transmembrane sialomucin Podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if Podocalyxin plays a functional role in breast tumor progression. Methods: MCF-7 breast cancer cells, which express little endogenous Podocalyxin, were stably transfected with wild type Podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous Podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting Podocalyxin for stable knockdown. In vitro, the effects of Podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Results: Forced overexpression of Podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, Podocalyxin overexpression induced a collective migration of MCF-7 tumor cell
Julie S Nielsen - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Open Access The cell surface mucin Podocalyxin regulates collective breast tumor budding
2016Co-Authors: Marcia L Graves, Kelly M. Mcnagny, Julie S Nielsen, Blake C Gilks, Pamela Austin, Erin M Bell, Jane A. Cipollone, Calvin D RoskelleyAbstract:Background: Overexpression of the transmembrane sialomucin Podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if Podocalyxin plays a functional role in breast tumor progression. Methods: MCF-7 breast cancer cells, which express little endogenous Podocalyxin, were stably transfected with wild type Podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous Podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting Podocalyxin for stable knockdown. In vitro, the effects of Podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Results: Forced overexpression of Podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, Podocalyxin overexpression induced a collective migration of MCF-7 tumor cell
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the cell surface mucin Podocalyxin regulates collective breast tumor budding
Breast Cancer Research, 2016Co-Authors: Marcia L Graves, Kelly M. Mcnagny, Julie S Nielsen, Jane Cipollone, Pamela Austin, Erin M Bell, C B Gilks, Calvin D RoskelleyAbstract:Overexpression of the transmembrane sialomucin Podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if Podocalyxin plays a functional role in breast tumor progression. MCF-7 breast cancer cells, which express little endogenous Podocalyxin, were stably transfected with wild type Podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous Podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting Podocalyxin for stable knockdown. In vitro, the effects of Podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Forced overexpression of Podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, Podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of Podocalyxin. In three-dimensional (3-D) culture, Podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous Podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture. Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.
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Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy
Breast Cancer Research, 2015Co-Authors: Kimberly Snyder, Michael R Hughes, Bradley John Hedberg, Jill Brandon, Diana Canals Hernaez, Peter J Bergqvist, Frederic Cruz, Marcia L Graves, Michelle E Turvey, Julie S NielsenAbstract:Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of Podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if Podocalyxin has a functional role in tumor progression. We silenced Podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed Podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how Podocalyxin affects tumorsphere formation in vitro and compared the ability of Podocalyxin-deficient and Podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman Podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. Although deletion of Podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing Podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that Podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate Podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman Podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. We show that Podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate Podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.
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the role of Podocalyxin in health and disease
Journal of The American Society of Nephrology, 2009Co-Authors: Julie S Nielsen, Kelly M. McnagnyAbstract:Podocalyxin, a sialomucin most closely related to CD34 and endoglycan, is expressed by kidney podocytes, hematopoietic progenitors, vascular endothelia, and a subset of neurons; aberrant expression has recently been implicated in a range of cancers. Through interactions with several intracellular proteins and at least one extracellular ligand, Podocalyxin regulates both adhesion and cell morphology. In the developing kidney, Podocalyxin plays an essential role in the formation and maintenance of podocyte foot processes, and its absence results in perinatal lethality. Podocalyxin expression in the hematopoietic system correlates with cell migration and the seeding of new hematopoietic tissues. In addition, it is abnormally expressed in subsets of breast, prostate, liver, pancreatic, and kidney cancer as well as leukemia. Strikingly, it is often associated with the most aggressive cases, and it is likely involved in metastasis. Thus, a thorough investigation of the normal activities of Podocalyxin may facilitate the development of new cancer treatment strategies.
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the cd34 related molecule Podocalyxin is a potent inducer of microvillus formation
PLOS ONE, 2007Co-Authors: Julie S Nielsen, Shierley Chelliah, Marcia L Graves, Calvin D Roskelley, Wayne A Vogl, Kelly M. McnagnyAbstract:Background Podocalyxin is a CD34-related transmembrane protein involved in hematopoietic cell homing, kidney morphogenesis, breast cancer progression, and epithelial cell polarization. Although this sialomucin has been shown to block cell adhesion, the mechanisms involved remain enigmatic. It has, however, been postulated that the adaptor proteins NHERF-1 and 2 could regulate apical targeting of Podocalyxin by linking it to the actin cytoskeleton. Principal Findings Here, in contrast, we find that full-length Podocalyxin acts to recruit NHERF-1 to the apical domain. Moreover, we show that ectopic expression of Podocalyxin in epithelial cells leads to microvillus formation along an expanded apical domain that extends laterally to the junctional complexes. Removal of the C-terminal PDZ-binding domain of Podocalyxin abolishes NHERF-1 recruitment but, surprisingly, has no effect on the formation of microvilli. Instead, we find that the extracellular domain and transmembrane region of Podocalyxin are sufficient to direct recruitment of filamentous actin and ezrin to the plasma membrane and induce microvillus formation. Conclusions/Significance Our data suggest that this single molecule can modulate NHERF localization and, independently, act as a key orchestrator of apical cell morphology, thereby lending mechanistic insights into its multiple roles as a polarity regulator, tumor progression marker, and anti-adhesin.
Calvin D Roskelley - One of the best experts on this subject based on the ideXlab platform.
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abstract 4911 characterizing the role of Podocalyxin s cytoplasmic tail domain in collective tumor invasion
Cancer Research, 2020Co-Authors: Erin M Bell, Kelly M. Mcnagny, Marcia L Graves, Pamela Dean, Calvin D RoskelleyAbstract:High expression of the single-pass transmembrane sialomucin, Podocalyxin, has been shown by many groups to correlate with poor disease outcome in a number of solid tumor types, including colorectal, ovarian, pancreatic and breast cancers. We had previously identified that high Podocalyxin expression in invasive ductal breast carcinoma was an independent indicator of poor survival (Somasiri et al., 2004, Cancer Res. 64:15). Further analysis of these tumor samples revealed that this decrease in survival occurred without the loss of membranous, junctional E-cadherin, suggesting that these tumors may have invaded collectively without necessitating an overt epithelial to mesenchymal transition (EMT). Experimentally, forced over-expression of Podocalyxin in polarity-disrupted human MCF7 breast cancer cells drives the formation of invasive orthotopic xenograft tumors and elongated, cohesive, and E-Cadherin-expressing spheroids in three-dimensional (3D) culture as compared to control (Graves et al., 2016, Breast Canc. Res. 18:11). Further, treatment of these Podocalyxin-overexpressing MCF7 cells with the myosin inhibitor, blebbistatin, and the small molecule inhibitor of ezrin-actin binding, NSC668394, resulted in decreased collective invasion and migration, respectively. Together these data suggest that Podocalyxin, through interactions with the actin cytoskeleton via its cytoplasmic tail binding partners, can facilitate increased collective epithelial tumor cell motility, at least in some contexts. To test this hypothesis, we generated Podocalyxin null MCF7 clones and cell populations using CRISPR-Cas9 genome editing and reconstituted these cells with mutant forms of Podocalyxin that are unable to interact with the scaffolding proteins NHERF and/or ezrin and hence with the actin cytoskeleton. Preliminary results from 3D culture and live imaging of these mutant Podocalyxin-expressing cells suggests that loss of Podocalyxin9s cytoplasmic tail results in decreased spheroid invasion that may be a result of deficiencies in actomyosin contractility. Hence, increased expression and mislocalization of Podocalyxin may facilitate aberrant interactions with the actin cytoskeleton and contractile machinery, driving enhanced cell motility and, in certain tumor microenvironments, promote collective tumor invasion. Citation Format: Erin M. Bell, Marcia L. Graves, Pamela Dean, Kelly M. McNagny, Calvin D. Roskelley. Characterizing the role of Podocalyxin9s cytoplasmic tail domain in collective tumor invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4911.
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distinct functional requirements for Podocalyxin in immature and mature podocytes reveal mechanisms of human kidney disease
Scientific Reports, 2020Co-Authors: Ido Refaeli, Michael R Hughes, Calvin D Roskelley, Wayne A Vogl, Alvin Kawai Wong, Mei Lin Z Bissonnette, Sean J Barbour, Benjamin S Freedman, Kelly M. McnagnyAbstract:Dominant and recessive mutations in Podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and Podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for Podocalyxin in all tissues (Podxl+/-). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl-/-), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/- mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/- mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which Podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/- mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.
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RESEARCH ARTICLE Open Access The cell surface mucin Podocalyxin regulates collective breast tumor budding
2016Co-Authors: Marcia L Graves, Kelly M. Mcnagny, Julie S Nielsen, Blake C Gilks, Pamela Austin, Erin M Bell, Jane A. Cipollone, Calvin D RoskelleyAbstract:Background: Overexpression of the transmembrane sialomucin Podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if Podocalyxin plays a functional role in breast tumor progression. Methods: MCF-7 breast cancer cells, which express little endogenous Podocalyxin, were stably transfected with wild type Podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous Podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting Podocalyxin for stable knockdown. In vitro, the effects of Podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Results: Forced overexpression of Podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, Podocalyxin overexpression induced a collective migration of MCF-7 tumor cell
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the cell surface mucin Podocalyxin regulates collective breast tumor budding
Breast Cancer Research, 2016Co-Authors: Marcia L Graves, Kelly M. Mcnagny, Julie S Nielsen, Jane Cipollone, Pamela Austin, Erin M Bell, C B Gilks, Calvin D RoskelleyAbstract:Overexpression of the transmembrane sialomucin Podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if Podocalyxin plays a functional role in breast tumor progression. MCF-7 breast cancer cells, which express little endogenous Podocalyxin, were stably transfected with wild type Podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous Podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting Podocalyxin for stable knockdown. In vitro, the effects of Podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. Forced overexpression of Podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, Podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of Podocalyxin. In three-dimensional (3-D) culture, Podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous Podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture. Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.
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The anti-adhesive mucin Podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma
Clinical & Experimental Metastasis, 2012Co-Authors: Jane A. Cipollone, Kelly M. Mcnagny, Marcia L Graves, Martin Köbel, Steve E. Kalloger, Tak Poon, C. Blake Gilks, Calvin D RoskelleyAbstract:High grade serous ovarian tumors often metastasize transperitoneally, a process that begins when small tumor nodules de-adhere and are released into the fluid of the abdominal cavity where they float freely to reach new sites on the peritoneal wall. Podocalyxin, a small anti-adhesive sialomucin, has been shown to contribute to non-adhesive membrane domain formation in some epithelia and is overexpressed in a variety of cancers. We therefore assessed Podocalyxin expression on a previously characterized tissue microarray and found that 87% (169/194) of high grade serous epithelial ovarian carcinomas were positive for Podocalyxin. In addition, cell surface localization of Podocalyxin was associated with a significant decrease in disease-free survival in these tumors. When Podocalyxin was force-expressed in serous ovarian carcinoma-derived OVCAR-3 cells it was targeted to the cell surface and it decreased the adhesion of these cells to mesothelial monolayers, fibronectin and immobilized β1 integrin-binding antibodies. This decrease in adhesion was associated with a modest decrease in cell surface β1 integrin. In monolayer culture, Podocalyxin was targeted to the free, apical domains of OVCAR-3 cells and it appeared to decrease β1 integrin levels on the attached basolateral domains of the same cells. Furthermore, in 3-dimensional basement membrane gel culture, the cells formed small, cohesive nodules and Podocalyxin localized to membrane domains at the cell–basement membrane interface. Therefore, Podocalyxin’s ability to facilitate the formation of non-adhesive membrane domains may contribute to the formation of free-floating high grade serous tumor nodules during the initial steps of transperitoneal metastasis.
David B. Kershaw - One of the best experts on this subject based on the ideXlab platform.
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Anuria, Omphalocele, and Perinatal Lethality in Mice Lacking the CD34-related Protein Podocalyxin
2013Co-Authors: Regis Doyonnas, David B. Kershaw, Christian Duhme, Helen Merkens, Shierley Chelliah, Thomas Graf, Kelly M. McnagnyAbstract:Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of Podocalyxin, we generated Podocalyxin-deficient (podxl �/ � ) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl �/ � mice, they fail to form foot processes and slit diaphragms and instead exhibit cell–cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl �/ � mice frequently display herniation of the gut (omphalocele), suggesting that Podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the Podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in Podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans. Key words: sialomucin • umbilical hernia • podocyte • hematopoiesis • vascular endothelium R. Doyonnas and D.B. Kershaw contributed equally to this manuscript
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overexpression of the anti adhesin Podocalyxin is an independent predictor of breast cancer progression
Cancer Research, 2004Co-Authors: Aruna Somasiri, David B. Kershaw, Julie S Nielsen, Nikita Makretsov, Marcia L Mccoy, Leah M Prentice, Blake C Gilks, Stephen Chia, Karen A Gelmon, David G HuntsmanAbstract:Podocalyxin is a CD34-related cell surface molecule with anti-adhesive qualities. We probed a tissue microarray (n = 272) linked to long-term outcome data and found that Podocalyxin was highly overexpressed in a distinct subset of invasive breast carcinomas (n = 15; 6%). Univariate disease-specific (P < 0.01) and multivariate regression (P < 0.0005) analyses indicated that this overexpression is an independent indicator of poor outcome. Forced Podocalyxin expression perturbed cell junctions between MCF-7 breast carcinoma cells, and it caused cell shedding from confluent monolayers. Therefore, Podocalyxin overexpression is a novel predictor of breast cancer progression that may contribute to the process by perturbing tumor cell adhesion.
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enhanced Podocalyxin expression alters the structure of podocyte basal surface
Journal of Cell Science, 2004Co-Authors: Constantinos G Economou, David B. Kershaw, Paraskevi V Kitsiou, Athina K Tzinia, Evridiki Panagopoulou, Evangelos Marinos, Dontscho Kerjaschki, Effie C TsilibaryAbstract:Glomerular basement membrane (GBM) and Podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of Podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of Podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-Podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus Podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of Podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-Podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing Podocalyxin, the presence of anti-Podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-β1 integrin antibody, thus Podocalyxin should compete with β1-related cell adhesion. We suggest that the observed Podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.
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human embryonal carcinoma tumor antigen gp200 gctm 2 is Podocalyxin
Biochemical and Biophysical Research Communications, 2003Co-Authors: Michael W Schopperle, David B. Kershaw, William C DewolfAbstract:We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as Podocalyxin. Protein sequence results of gp200 peptides match with Podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for Podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of Podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of Podocalyxin expression on malignant cells.
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pdz domain mediated interaction of rabbit Podocalyxin and na h exchange regulatory factor 2
American Journal of Physiology-renal Physiology, 2002Co-Authors: Samuel W Straight, David B. KershawAbstract:The transmembrane sialoglycoprotein Podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-Hi...
William C Dewolf - One of the best experts on this subject based on the ideXlab platform.
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the human cancer and stem cell marker Podocalyxin interacts with the glucose 3 transporter in malignant pluripotent stem cells
Biochemical and Biophysical Research Communications, 2010Co-Authors: William M Schopperle, Jung M Lee, William C DewolfAbstract:Podocalyxin, an integral plasma membrane cell-adhesion glycoprotein, is a marker of human pluripotent and multipotent stem cells. Podocalyxin is also a marker of many types of cancers and its expression correlates with an aggressive and poor-prognosis tumor phenotype. The function of Podocalyxin in stem cells and malignant cells is unknown. Protein sequence data obtained from purified Podocalyxin protein isolated from embryonal carcinoma cancer stem cells reveals peptide sequence data for the glucose-3-transporter. Protein-precipitation experiments of embryonal carcinoma protein extracts identify a Podocalyxin/glucose-3-transporter protein complex. Cell imaging studies demonstrate co-localization of Podocalyxin and glucose-3-transporter and confirm the interaction in vivo. Finally, siRNA Podocalyxin-knockdown experiments show decreased expression levels of the glucose-3-transporter. These findings suggest a novel interaction of the glucose-3-transporter and the cell-adhesion protein Podocalyxin. In pluripotent stem cells and in human cancer disease, Podocalyxin may function in part to regulate and maintain the cell surface expression of the glucose-3-transporter.
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the tra 1 60 and tra 1 81 human pluripotent stem cell markers are expressed on Podocalyxin in embryonal carcinoma
Stem Cells, 2007Co-Authors: William M Schopperle, William C DewolfAbstract:We have previously identified the cell adhesion protein Podocalyxin expressed in a human pluripotent stem cell, embryonal carcinoma (EC), which is a malignant germ cell. Podocalyxin is a heavily glycosylated membrane protein with amino acid sequence homology to the hematopoietic stem cell marker CD34. Since the initial discovery of Podocalyxin in a cancerous stem cell, numerous new studies have identified Podocalyxin in many different human cancers and in embryonic stem cells lines (ES) derived from human embryos. Embryonal carcinoma, as do all human pluripotent stem cells, expresses TRA-1-60 and TRA-1-81 antigens, and although their molecular identities are unknown, they are commonly used as markers of undifferentiated pluripotent human stem cells. We report here that purified Podocalyxin from embryonal carcinoma has binding activity with the TRA-1-60 and TRA-1-81 antibodies. Embryonal carcinoma cells treated with retinoic acid undergo differentiation and lose the TRA-1-60/TRA-1-81 markers from their plasma membrane surface. We show that Podocalyxin is modified in the retinoic acid-treated cells and has an apparent molecular mass of 170 kDa on protein blots as compared with the apparent 200-kDa molecular weight form of Podocalyxin expressed in untreated cells. Furthermore, the modified form of Podocalyxin no longer reacts with the TRA-1-60/TRA-1-81 antibodies. Thus, embryonal carcinoma expresses two distinct forms of Podocalyxin, and the larger version is a molecular carrier of the human stem cell-defining antigens TRA-1-60 and TRA-1-81.
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human embryonal carcinoma tumor antigen gp200 gctm 2 is Podocalyxin
Biochemical and Biophysical Research Communications, 2003Co-Authors: Michael W Schopperle, David B. Kershaw, William C DewolfAbstract:We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as Podocalyxin. Protein sequence results of gp200 peptides match with Podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for Podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of Podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of Podocalyxin expression on malignant cells.
