Constantinos Mikelis - Academia.edu (original) (raw)
Papers by Constantinos Mikelis
International Journal of Cancer, Jun 1, 2023
Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP... more Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165) and pleiotrophin (PTN). It is also over or under‐expressed in various tumor types. In this study, we used genetically engineered Ptprz1−/− and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1−/− lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1−/− compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane‐treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (β3) integrin is decreased in Ptprz1−/− LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c‐Met tyrosine kinase (TK) and Akt kinase activities. However, only c‐Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1−/− mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c‐Met and Akt in a PTPRZ1‐dependent manner in endothelial cells, and their stimulatory effects are abolished by the c‐Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c‐Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c‐Met activation by VEGFA and PTN.
Current Cancer Drug Targets, Sep 1, 2004
Pharmaceutics, Nov 26, 2022
Anaplastic lympoma kinase (ALK) has been initially identified in anaplastic large-cell lymphomas ... more Anaplastic lympoma kinase (ALK) has been initially identified in anaplastic large-cell lymphomas in the form of fusion proteins. In lung cancer, 5-10% of patients present with EML4-ALK fusion genes. No involvement of wild-type ALK has been ever reported in lung cancer. In vitro data have suggested that ALK interacts with receptor protein tyrosine phosphatase zeta 1 (PTPRZ1) and the latter inhibits ALK tyrosine kinase (TK) activity. PTPRZ1 has been associated with various types of cancer, but its role in lung adenocarcinoma has not been studied. Here, we show that the ALK TK inhibitor (TKI) crizotinib abolished the increased lung carcinogenesis, angiogenesis and macrophage infiltration observed in Ptprz1-/- mice, with minimal effect in Ptprz1+/+ mice. Wild-type ALK phosphorylation and downstream signaling was enhanced in Ptprz1-/- tumors and lung microvascular endothelial cells (LMVECs). Genetic or pharmacological deletion of PTPRZ1 in LMVECs results in enhanced proliferation, migration, and tube formation in vitro, effects abolished by selective ALK TKIs. In human lung adenocarcinoma cells in vitro, low expression of PTPRZ1 correlates with K-Ras mutations and intense aggressiveness of the cancer cells and crizotinib abolishes this effect. Furthermore, low PTPRZ1 expression in human patient lung adenocarcinomas correlates with poor survival, while smokers or patients with K-Ras mutations have decreased PTPRZ1 expression and worse prognosis. All together these data suggest that low PTPRZ1 expression may act as an important biomarker for adenocarcinoma patients’ prognosis and responsiveness to ALK TKIs.
Methods in molecular biology, Aug 18, 2020
The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasio... more The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as "bait" to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.
Methods in molecular biology, Aug 18, 2020
American Journal of Physiology-heart and Circulatory Physiology, 2022
Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and s... more Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and seems to affect heart development. In both mouse and zebrafish animal models, loss of PTPRZ1 results in dilated left ventricle cavity, decreased ejection fraction, and fraction shortening, with no signs of cardiac hypertrophy. PTPRZ1 also seems to be involved in atrioventricular canal specification, outflow tract morphogenesis, and heart angiogenesis. These results suggest that PTPRZ1 plays a role in heart development and support the hypothesis that it may be involved in congenital cardiac pathologies.
International Journal of Molecular Sciences, Jan 23, 2020
Angiogenesis, Feb 1, 2022
Lymphangiogenesis is an essential physiological process but also a determining factor in vascular... more Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.
Methods in molecular biology, 2019
Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation... more Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation. Physiologically, angiogenesis is more prominent during development and wound healing and its dysregulation drives or is related to several diseases, including cancer. The endothelial cells are the main regulators of the angiogenic process, and thus the angiogenic outcome is assessed based on the effect on endothelial cell functions. Several in vitro and in vivo techniques have been developed to assess the effect of various factors on angiogenesis. Compared to the in vivo techniques, the in vitro techniques are considered less physiologically relevant. This has been partially overcome by the development of 3-dimensional (3D) in vitro models, one of which is the spheroid assay or 3D sprouting assay that exploits the effect of the extracellular matrix to endothelial cell functions. This chapter focuses on the description of the spheroid assay and mentions the variations and potential applications this assay can have.
Tumor Biology, Jun 1, 2017
Nature Communications, Apr 10, 2015
Scientific Reports, Aug 12, 2019
Springer eBooks, Dec 19, 2022
Scientific Reports, Mar 22, 2021
The FASEB Journal
G protein coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G... more G protein coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation. Among them, PDZ‐RhoGEF, leukemia‐associated Rho GEF (LARG) and p115‐RhoGEF (p115) share the presence of a regulator of G protein signaling‐homology (RGS) domain. There is limited information on the biological roles of this RGS‐containing family of RhoGEFs in vivo. Here, we investigated the biological role of two structurally related RhoGEFs: PDZ‐RhoGEF and LARG, by generating knockout mice. Although these mice do not display any overt phenotype, compound deficiency in PDZ‐RhoGEF and LARG leads to early embryonic lethality and to complex developmental defects. Signaling from Gα11/q‐linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all 3 RGS‐containing ...
Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous... more Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation....
International Journal of Cancer, Jun 1, 2023
Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP... more Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165) and pleiotrophin (PTN). It is also over or under‐expressed in various tumor types. In this study, we used genetically engineered Ptprz1−/− and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1−/− lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1−/− compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane‐treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (β3) integrin is decreased in Ptprz1−/− LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c‐Met tyrosine kinase (TK) and Akt kinase activities. However, only c‐Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1−/− mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c‐Met and Akt in a PTPRZ1‐dependent manner in endothelial cells, and their stimulatory effects are abolished by the c‐Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c‐Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c‐Met activation by VEGFA and PTN.
Current Cancer Drug Targets, Sep 1, 2004
Pharmaceutics, Nov 26, 2022
Anaplastic lympoma kinase (ALK) has been initially identified in anaplastic large-cell lymphomas ... more Anaplastic lympoma kinase (ALK) has been initially identified in anaplastic large-cell lymphomas in the form of fusion proteins. In lung cancer, 5-10% of patients present with EML4-ALK fusion genes. No involvement of wild-type ALK has been ever reported in lung cancer. In vitro data have suggested that ALK interacts with receptor protein tyrosine phosphatase zeta 1 (PTPRZ1) and the latter inhibits ALK tyrosine kinase (TK) activity. PTPRZ1 has been associated with various types of cancer, but its role in lung adenocarcinoma has not been studied. Here, we show that the ALK TK inhibitor (TKI) crizotinib abolished the increased lung carcinogenesis, angiogenesis and macrophage infiltration observed in Ptprz1-/- mice, with minimal effect in Ptprz1+/+ mice. Wild-type ALK phosphorylation and downstream signaling was enhanced in Ptprz1-/- tumors and lung microvascular endothelial cells (LMVECs). Genetic or pharmacological deletion of PTPRZ1 in LMVECs results in enhanced proliferation, migration, and tube formation in vitro, effects abolished by selective ALK TKIs. In human lung adenocarcinoma cells in vitro, low expression of PTPRZ1 correlates with K-Ras mutations and intense aggressiveness of the cancer cells and crizotinib abolishes this effect. Furthermore, low PTPRZ1 expression in human patient lung adenocarcinomas correlates with poor survival, while smokers or patients with K-Ras mutations have decreased PTPRZ1 expression and worse prognosis. All together these data suggest that low PTPRZ1 expression may act as an important biomarker for adenocarcinoma patients’ prognosis and responsiveness to ALK TKIs.
Methods in molecular biology, Aug 18, 2020
The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasio... more The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as "bait" to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.
Methods in molecular biology, Aug 18, 2020
American Journal of Physiology-heart and Circulatory Physiology, 2022
Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and s... more Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and seems to affect heart development. In both mouse and zebrafish animal models, loss of PTPRZ1 results in dilated left ventricle cavity, decreased ejection fraction, and fraction shortening, with no signs of cardiac hypertrophy. PTPRZ1 also seems to be involved in atrioventricular canal specification, outflow tract morphogenesis, and heart angiogenesis. These results suggest that PTPRZ1 plays a role in heart development and support the hypothesis that it may be involved in congenital cardiac pathologies.
International Journal of Molecular Sciences, Jan 23, 2020
Angiogenesis, Feb 1, 2022
Lymphangiogenesis is an essential physiological process but also a determining factor in vascular... more Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.
Methods in molecular biology, 2019
Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation... more Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation. Physiologically, angiogenesis is more prominent during development and wound healing and its dysregulation drives or is related to several diseases, including cancer. The endothelial cells are the main regulators of the angiogenic process, and thus the angiogenic outcome is assessed based on the effect on endothelial cell functions. Several in vitro and in vivo techniques have been developed to assess the effect of various factors on angiogenesis. Compared to the in vivo techniques, the in vitro techniques are considered less physiologically relevant. This has been partially overcome by the development of 3-dimensional (3D) in vitro models, one of which is the spheroid assay or 3D sprouting assay that exploits the effect of the extracellular matrix to endothelial cell functions. This chapter focuses on the description of the spheroid assay and mentions the variations and potential applications this assay can have.
Tumor Biology, Jun 1, 2017
Nature Communications, Apr 10, 2015
Scientific Reports, Aug 12, 2019
Springer eBooks, Dec 19, 2022
Scientific Reports, Mar 22, 2021
The FASEB Journal
G protein coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G... more G protein coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation. Among them, PDZ‐RhoGEF, leukemia‐associated Rho GEF (LARG) and p115‐RhoGEF (p115) share the presence of a regulator of G protein signaling‐homology (RGS) domain. There is limited information on the biological roles of this RGS‐containing family of RhoGEFs in vivo. Here, we investigated the biological role of two structurally related RhoGEFs: PDZ‐RhoGEF and LARG, by generating knockout mice. Although these mice do not display any overt phenotype, compound deficiency in PDZ‐RhoGEF and LARG leads to early embryonic lethality and to complex developmental defects. Signaling from Gα11/q‐linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all 3 RGS‐containing ...
Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous... more Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation....