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Klaus Wielckens - One of the best experts on this subject based on the ideXlab platform.
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a critical role for collagen ii in cartilage Matrix Degradation collagen ii induces pro inflammatory cytokines and mmps in primary human chondrocytes
Journal of Orthopaedic Research, 2009Co-Authors: Andreas R Klatt, Brigitte Paulklausch, Gabriele Klinger, Getrud Kuhn, Joerg H Renno, Marc Banerjee, Gebhart Malchau, Klaus WielckensAbstract:We report a process that results in the acceleration of Matrix Degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and Matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1β. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IκB concentration were monitored by immunoblot analysis to detect NFκB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1β, IL-6, and IL-8. At the same time, inhibition of p38 and IκB Degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFκB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage Matrix Degradation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:65–70, 2009
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a critical role for collagen ii in cartilage Matrix Degradation collagen ii induces pro inflammatory cytokines and mmps in primary human chondrocytes
Journal of Orthopaedic Research, 2009Co-Authors: Andreas R Klatt, Brigitte Paulklausch, Gabriele Klinger, Getrud Kuhn, Joerg H Renno, Marc Banerjee, Gebhart Malchau, Klaus WielckensAbstract:We report a process that results in the acceleration of Matrix Degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and Matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1beta. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IkappaB concentration were monitored by immunoblot analysis to detect NFkappaB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1beta, IL-6, and IL-8. At the same time, inhibition of p38 and IkappaB Degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFkappaB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage Matrix Degradation.
Jeffrey E Segall - One of the best experts on this subject based on the ideXlab platform.
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tc10 regulates breast cancer invasion and metastasis by controlling membrane type 1 Matrix metalloproteinase at invadopodia
Unknown Journal, 2020Co-Authors: Maren Hulsemann, Jeffrey E Segall, Sara K Donnelly, Polina V Verkhusha, Serena P H Mao, Louis HodgsonAbstract:Abstract During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular Matrix Degradation required for the success of the invasive cascade. In this study, we demonstrated that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 Matrix metalloproteinase (MT1-MMP)-driven extracellular Matrix Degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at invadopodia. By utilizing our new Forster resonance energy transfer (FRET) biosensor, we demonstrated the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates TC10 activity and function at invadopodia through the activation of p190RhoGAP and the downstream interacting effector Exo70 at the invadopodia sites. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10, on the invasive potential of breast cancer cells during invasion and metastasis.
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rac3 regulates breast cancer invasion and metastasis by controlling adhesion and Matrix Degradation
Journal of Cell Biology, 2017Co-Authors: Sara K Donnelly, Jeffrey E Segall, John S Condeelis, Jose Javier Bravocordero, Serena P H Mao, Ramon M Cabrera, John R Christin, Wenjun Guo, Louis HodgsonAbstract:The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the Matrix Degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular Matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of Matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for Matrix Degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.
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microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Archives of Pathology & Laboratory Medicine, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:CONTEXT: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. OBJECTIVE: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. DESIGN: For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. RESULTS: Increased miR-375 expression in HNSCC cells suppresses extracellular Matrix Degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. CONCLUSIONS: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.
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abstract 185 microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Cancer Research, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:Context.— Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. Objective.— To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. Design.— For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of...
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abstract 1452 microrna 375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity
Cancer Research, 2014Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas M. Harris, John S Condeelis, Ruth Hogue Angeletti, Jeffrey E SegallAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group previously reported that miR-375 expression levels correlate with patient survival, recurrence and distant metastasis. We have previously observed diminished HNSCC invasion in vitro correlates with increased miR-375 expression. To assess whether the miR-375 over-expressing transductant lines have reduced Matrix Degradation properties, a fluorescent Matrix Degradation assay was used. We have observed that miR-375 over-expression in HNSCC cells suppresses extracellular Matrix Degradation. We further determined whether miR-375 over-expressing cells show reduced invadopodium formation and maturation. We observed miR-375 expression in HNSCC cell lines reduces the number of mature invadopodia, but does not affect levels of invadopodium precursors. We examined the phosphorylation and total levels of key invadopodia components (Tks5 and cortactin) to determine whether changes in regulation or expression of these components may lead impaired Matrix Degradation properties. We determined that miR-375 expression in HNSCC cell lines does not reduce cellular levels of cortactin and Tks5, nor is tyrosine phosphorylation of cortactin altered. To identify candidate proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC). Candidate target proteins are being validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, Jihyeon Lim, Ruth Angeletti, John Condeelis, Thomas Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall. MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2014-1452
John S Condeelis - One of the best experts on this subject based on the ideXlab platform.
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myosin iia heavy chain phosphorylation on s1943 regulates tumor metastasis
Experimental Cell Research, 2018Co-Authors: Laura Norwood E Toro, John S Condeelis, Yarong Wang, Joan G Jones, Jonathan M Backer, Anne R BresnickAbstract:Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for Matrix Degradation in vitro and experimental metastasis in vivo. These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular Matrix Degradation and tumor cell invasion and metastasis.
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rac3 regulates breast cancer invasion and metastasis by controlling adhesion and Matrix Degradation
Journal of Cell Biology, 2017Co-Authors: Sara K Donnelly, Jeffrey E Segall, John S Condeelis, Jose Javier Bravocordero, Serena P H Mao, Ramon M Cabrera, John R Christin, Wenjun Guo, Louis HodgsonAbstract:The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the Matrix Degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular Matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of Matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for Matrix Degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.
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phosphorylated cortactin recruits vav2 guanine nucleotide exchange factor to activate rac3 and promote invadopodial function in invasive breast cancer cells
Molecular Biology of the Cell, 2017Co-Authors: Brian Rosenberg, John S Condeelis, Christopher C Mader, Hava Gilhenn, Tiffany L Halo, Taofei Yin, Kazuya Machida, Anthony J KoleskeAbstract:Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular Matrix. Phosphorylation of the actin nucleation-promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor-Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient Matrix Degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, Matrix Degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces Matrix Degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.
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microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Archives of Pathology & Laboratory Medicine, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:CONTEXT: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. OBJECTIVE: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. DESIGN: For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. RESULTS: Increased miR-375 expression in HNSCC cells suppresses extracellular Matrix Degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. CONCLUSIONS: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.
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abstract 185 microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Cancer Research, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:Context.— Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. Objective.— To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. Design.— For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of...
Andreas R Klatt - One of the best experts on this subject based on the ideXlab platform.
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a critical role for collagen ii in cartilage Matrix Degradation collagen ii induces pro inflammatory cytokines and mmps in primary human chondrocytes
Journal of Orthopaedic Research, 2009Co-Authors: Andreas R Klatt, Brigitte Paulklausch, Gabriele Klinger, Getrud Kuhn, Joerg H Renno, Marc Banerjee, Gebhart Malchau, Klaus WielckensAbstract:We report a process that results in the acceleration of Matrix Degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and Matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1β. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IκB concentration were monitored by immunoblot analysis to detect NFκB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1β, IL-6, and IL-8. At the same time, inhibition of p38 and IκB Degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFκB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage Matrix Degradation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:65–70, 2009
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a critical role for collagen ii in cartilage Matrix Degradation collagen ii induces pro inflammatory cytokines and mmps in primary human chondrocytes
Journal of Orthopaedic Research, 2009Co-Authors: Andreas R Klatt, Brigitte Paulklausch, Gabriele Klinger, Getrud Kuhn, Joerg H Renno, Marc Banerjee, Gebhart Malchau, Klaus WielckensAbstract:We report a process that results in the acceleration of Matrix Degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and Matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1beta. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IkappaB concentration were monitored by immunoblot analysis to detect NFkappaB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1beta, IL-6, and IL-8. At the same time, inhibition of p38 and IkappaB Degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFkappaB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage Matrix Degradation.
Ved P Sharma - One of the best experts on this subject based on the ideXlab platform.
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microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Archives of Pathology & Laboratory Medicine, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:CONTEXT: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. OBJECTIVE: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. DESIGN: For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. RESULTS: Increased miR-375 expression in HNSCC cells suppresses extracellular Matrix Degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. CONCLUSIONS: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.
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abstract 185 microrna 375 suppresses extracellular Matrix Degradation and invadopodial activity in head and neck squamous cell carcinoma
Cancer Research, 2015Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas J Ow, Thomas M. Harris, John S Condeelis, Jeffrey E SegallAbstract:Context.— Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated Degradation of the extracellular Matrix. Objective.— To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. Design.— For evaluation of the Matrix Degradation properties of the HNSCC lines, an invadopodial Matrix Degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of...
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abstract 1452 microrna 375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity
Cancer Research, 2014Co-Authors: Lizandra Jimenez, Ved P Sharma, Geoffrey Childs, Michael B Prystowsky, Thomas M. Harris, John S Condeelis, Ruth Hogue Angeletti, Jeffrey E SegallAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group previously reported that miR-375 expression levels correlate with patient survival, recurrence and distant metastasis. We have previously observed diminished HNSCC invasion in vitro correlates with increased miR-375 expression. To assess whether the miR-375 over-expressing transductant lines have reduced Matrix Degradation properties, a fluorescent Matrix Degradation assay was used. We have observed that miR-375 over-expression in HNSCC cells suppresses extracellular Matrix Degradation. We further determined whether miR-375 over-expressing cells show reduced invadopodium formation and maturation. We observed miR-375 expression in HNSCC cell lines reduces the number of mature invadopodia, but does not affect levels of invadopodium precursors. We examined the phosphorylation and total levels of key invadopodia components (Tks5 and cortactin) to determine whether changes in regulation or expression of these components may lead impaired Matrix Degradation properties. We determined that miR-375 expression in HNSCC cell lines does not reduce cellular levels of cortactin and Tks5, nor is tyrosine phosphorylation of cortactin altered. To identify candidate proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC). Candidate target proteins are being validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, Jihyeon Lim, Ruth Angeletti, John Condeelis, Thomas Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall. MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2014-1452
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talin regulates moesin nhe 1 recruitment to invadopodia and promotes mammary tumor metastasis
Journal of Cell Biology, 2014Co-Authors: Brian T Beaty, Ved P Sharma, Jose Javier Bravocordero, Louis Hodgson, Yarong Wang, Veronika Miskolci, John S CondeelisAbstract:Invadopodia are actin-rich protrusions that degrade the extracellular Matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial Matrix Degradation and three-dimensional extracellular Matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated Matrix Degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin–NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and Matrix Degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.
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β1 integrin regulates arg to promote invadopodial maturation and Matrix Degradation
Molecular Biology of the Cell, 2013Co-Authors: Brian T Beaty, Ved P Sharma, Jose Javier Bravocordero, Anthony J Koleske, Robert J Eddy, Mark A Simpson, John S CondeelisAbstract:β1 integrin has been shown to promote metastasis in a number of tumor models, including breast, ovarian, pancreatic, and skin cancer; however, the mechanism by which it does so is poorly understood. Invasive membrane protrusions called invadopodia are believed to facilitate extracellular Matrix Degradation and intravasation during metastasis. Previous work showed that β1 integrin localizes to invadopodia, but its role in regulating invadopodial function has not been well characterized. We find that β1 integrin is required for the formation of mature, Degradation-competent invadopodia in both two- and three-dimensional matrices but is dispensable for invadopodium precursor formation in metastatic human breast cancer cells. β1 integrin is activated during invadopodium precursor maturation, and forced β1 integrin activation enhances the rate of invadopodial Matrix proteolysis. Furthermore, β1 integrin interacts with the tyrosine kinase Arg and stimulates Arg-dependent phosphorylation of cortactin on tyrosine 421. Silencing β1 integrin with small interfering RNA completely abrogates Arg-dependent cortactin phosphorylation and cofilin-dependent barbed-end formation at invadopodia, leading to a significant decrease in the number and stability of mature invadopodia. These results describe a fundamental role for β1 integrin in controlling actin polymerization–dependent invadopodial maturation and Matrix Degradation in metastatic tumor cells.