Extracellular Matrix Degradation Activity Research Articles

Are lower levels of apoptosis and autophagy behind adenomyotic lesion survival?

Research questionHow are markers of cell death, invasiveness and progesterone signalling expressed in endometrium and ectopic lesions from adenomyosis patients? DesignFormalin-fixed paraffin-embedded tissue was collected from 15 control and 15 adenomyosis participants . To assess cell survival capacity, caspase 3 and microtubule-associated proteins 1A/1B light chain 3B (LC3B) were immunolabelled as markers of apoptosis and autophagy respectively. Matrix metalloproteinase 9 (MMP9) expression served as a marker of extracellular matrix degradation and invasion activity. Progesterone receptors were immunostained to detect evidence of progesterone resistance. ResultsCaspase 3 expression was significantly lower in the stromal (P = 0.0013) and epithelial (P = 0.0157) compartments of adenomyotic lesions than in healthy endometrial tissue. In the stroma, caspase 3 expression was significantly weaker in lesions than in corresponding eutopic endometrium (P = 0.0006). LC3B immunostaining was significantly decreased in adenomyotic stroma compared with corresponding eutopic endometrium (P = 0.0349). A significantly higher expression of MMP9 was detected in eutopic stroma from adenomyosis patients than in healthy tissue (P = 0.0295). Progesterone receptor immunostaining was found to be significantly weaker in the stroma of endometrium and ectopic lesions from adenomyosis patients than disease-free women (P = 0.0001; P = 0.0021). ConclusionsAdenomyotic lesions show lower levels of apoptosis and autophagy, suggesting that aberrant cell survival may be involved in disease pathogenesis. MMP9 appears to contribute to endometrial invasiveness in adenomyosis, as its expression is more pronounced in endometrium from these women than women without the disease. Evidence of progesterone resistance can be found in endometrium and ectopic lesions from adenomyosis patients, and may drive disease development and account for the failure of certain patients to respond to progestogens.

Midline 1 interacting protein 1 promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in HCC.

Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis. Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities. Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression.

Rap1b-loss increases neutrophil lactate dehydrogenase activity to enhance neutrophil migration and acute inflammation in vivo.

Neutrophils are critical for host immune defense; yet, aberrant neutrophil tissue infiltration triggers tissue damage. Neutrophils are heterogeneous functionally, and adopt 'normal' or 'pathogenic' effector function responses. Understanding neutrophil heterogeneity could provide specificity in targeting inflammation. We previously identified a signaling pathway that suppresses neutrophilmediated inflammation via integrin-mediated Rap1b signaling pathway. Here, we used Rap1-deficient neutrophils and proteomics to identify pathways that specifically control pathogenic neutrophil effector function. We show neutrophil acidity is normally prevented by Rap1b during normal immune response with loss of Rap1b resulting in increased neutrophil acidity via enhanced Ldha activity and abnormal neutrophil behavior. Acidity drives the formation of abnormal invasive-like protrusions in neutrophils, causing a shift to transcellular migration through endothelial cells. Acidity increases neutrophil extracellular matrix degradation activity and increases vascular leakage in vivo. Pathogenic inflammatory condition of ischemia/reperfusion injury is associated with increased neutrophil transcellular migration and vascular leakage. Reducing acidity with lactate dehydrogenase inhibition in vivo limits tissue infiltration of pathogenic neutrophils but less so of normal neutrophils, and reduces vascular leakage. Acidic milieu renders neutrophils more dependent on Ldha activity such that their effector functions are more readily inhibited by small molecule inhibitor of Ldha activity, which offers a therapeutic window for antilactate dehydrogenase treatment in specific targeting of pathogenic neutrophils in vivo.

Abstract 5067: Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function

Abstract Malregulation of signal transduction events controlling cortical actin cytoskeleton dynamics is responsible for to enhancing tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in many cancers, corresponding with increased tumor progression and poor patient outcome. In many cancer types, elevated Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. While cortactin-mediated activation of Arp2/3 complex has been well established, the objective of this study is to identify upstream signaling pathways responsible for modulating the interaction between cortactin and Arp2/3 complex in invasive carcinoma cells. We have determined that casein kinase (CK) 2α is responsible for disrupting the ability of cortactin to bind and/or activate Arp2/3 complex. CK2α directly phosphorylates cortactin at a conserved threonine 24 (T24) adjacent to the canonical Arp2/3 binding motif in the cortactin amino-terminal acidic (NTA) domain. CK2α phosphorylation of cortactin T24 impairs the ability of cortactin to activate Arp2/3 actin nucleation as determined by spectrofluorometric analysis. Decreased invadopodia formation and ECM degradation activity is observed in head and neck squamous cell carcinoma (HNSCC) cells with shRNA-mediated CK2α knockdown, expression of a CK2α phosphorylation-null cortactin mutant (T24A), and with treatment of the Phase I CK2α inhibitor CX-4945 (Silmitasertib). Collectively, these data suggest that CK2α-mediated cortactin phosphorylation at T24 is a critical event in regulating the interaction with Arp2/3 complex, identifying a key anti-invasive mechanism impacted by targeted anti-CK2α therapeutics. Citation Format: Steven M. Markwell, Amanda Gatesman Ammer, Erik T. Interval, Dorothy A. Schafer, River A. Hames, Scott A. Weed. Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5067.

Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma.

The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer.

The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based WAVE2 complex.

BackgroundAbl interactor (Abi) family proteins play significant roles in actin cytoskeleton organization through participation in the WAVE complex. Mammals possess three Abi proteins: Abi-1, Abi-2, and NESH/Abi-3. Abi-1 and Abi-2 were originally identified as Abl tyrosine kinase-binding proteins. It has been disclosed that Abi-1 acts as a bridge between c-Abl and WAVE2, and c-Abl-mediated WAVE2 phosphorylation promotes actin remodeling. We showed previously that NESH/Abi-3 is present in the WAVE2 complex, but neither binds to c-Abl nor promotes c-Abl-mediated phosphorylation of WAVE2.ResultsIn this study, we characterized NESH/Abi-3 in more detail, and compared its properties with those of Abi-1 and Abi-2. NESH/Abi-3 was ectopically expressed in NIH3T3 cells, in which Abi-1, but not NESH/Abi-3, is expressed. The expression of NESH/Abi-3 caused degradation of endogenous Abi-1, which led to the formation of a NESH/Abi-3-based WAVE2 complex. When these cells were plated on fibronectin-coated dishes, the translocation of WAVE2 to the plasma membrane was significantly reduced and the formation of peripheral lamellipodial structures was disturbed, suggesting that the NESH/Abi-3-based WAVE2 complex was unable to help produce lamellipodial protrusions. Next, Abi-1, Abi-2, or NESH/Abi-3 was expressed in v-src-transformed NIH3T3 cells. Only in NESH/Abi-3-expressed cells did treatment with an Abl kinase inhibitor, imatinib mesylate, or siRNA-mediated knockdown of c-Abl promote the formation of invadopodia, which are ventral membrane protrusions with extracellular matrix degradation activity. Structural studies showed that a linker region between the proline-rich regions and the Src homology 3 (SH3) domain of Abi-1 is crucial for its interaction with c-Abl and c-Abl-mediated phosphorylation of WAVE2.ConclusionsThe NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based one, and NESH/Abi-3 may be involved in the formation of ventral protrusions under certain conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0119-5) contains supplementary material, which is available to authorized users.

Abstract 185: MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer having an association with locoregional recurrence and lymph node metastasis. We previously reported that patients with lower microRNA-375 (miR-375) tumor:normal expression levels showed significantly decreased disease-specific survival, increased locoregional recurrence and distant metastasis. We also previously described that HNSCC cells with increased miR-375 showed significantly diminished invasion in vitro. The ability of cancer cells to degrade extracellular matrix (ECM) through secreted and membrane-bound proteases allows local invasion into the surrounding stroma, as well as possible metastasis. Invadopodia are specialized actin-rich structures, which mediate ECM degradation. Our objective was to determine whether elevated miR-375 expression in HNSCC cells also affects invadopodia formation and activity. For the evaluation of the matrix degradation properties of UMSCC1 and OSC19 cell lines, an invadopodial matrix degradation assay was used. Western blot analyses were conducted to measure the total protein levels of selected invadopodia-associated proteins. The tyrosine phosphorylation states of cortactin were evaluated by immunoprecipitation experiments. For the detection of levels of secreted proteases in the conditioned cell growth medium, Human Protease Arrays were used. Quantitative RT-PCR measurements were used to evaluate the mRNA expression levels of the commonly regulated proteases. We observed that the HNSCC cell lines with elevated miR-375 expression showed significant reductions in ECM degradation. We further identified that HNSCC cells expressing increased miR-375 expression had significantly suppressed amounts of mature invadopodia, although the levels of invadopodium precursors were not significantly altered. We determined that increased miR-375 expression in the HNSCC cell lines did not reduce cellular levels of invadopodia components, such as Tks5, cortactin and fascin. We also did not observe significant alterations to the tyrosine phosphorylation states of cortactin in the HNSCC cell lines. However, we found that the HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression and secreted levels of specific proteases, including Kallikrein 6, Kallikrein 10, and MMP-9. In summary, we have extended our understanding of the impact of miR-375 expression levels on HNSCC invasion. We demonstrated that increased miR-375 expression in HNSCC cells reduced ECM degradation and invadopodial activity. Our data suggest that reduced miR-375 expression in HNSCC patients can contribute to the invasive properties of head and neck cancer through increased invadopodial activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, John S. Condeelis, Thomas M. Harris, Thomas J. Ow, Michael B. Prystowsky, Geoffrey J. Childs, Jeffrey E. Segall. MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 185. doi:10.1158/1538-7445.AM2015-185

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma.

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. To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. 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. 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. MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.

Abstract 3796: Endophilin II promotes breast tumor progression and metastasis.

Abstract Invadopodia are filamentous actin-based membrane protrusions with extracellular matrix (ECM) degrading activity that are implicated in tumor progression and metastasis. A key step in invadopodia formation involves the recruitment of the membrane type 1 matrix metalloprotease (MT1-MMP) to invadopodia precursors. Surface expression of MT1-MMP promotes ECM degradation and cell invasiveness, and this is dependent on Src kinase-induced phosphorylation and inhibition of endocytic proteins. In fibroblasts, MT1-MMP internalization was regulated by Endophilin II (Endo II), a widely expressed protein implicated in synaptic vesicle uncoating and recycling in neurons. Here, we tested the role of Endo II in invadopodia formation in basal-like breast cancer cells, and in tumor progression and metastasis in mice. High levels of Endo II expression were observed in basal breast cancer cell lines with high Src kinase and ECM-degrading activity. Immunohistochemistry staining also identified Endo II expression in primary breast tumors and lymph node metastases. To define the functions of Endo II, we achieved stable silencing of Endo II in two basal breast cancer cell lines (MDA-MB-231, HCC1806). Although Endo II was not required for uptake of receptors via clathrin-mediated endocytosis (EGFR, TfR), we observed a significant reduction in numbers of invadopodia and ECM degradation upon Endo II silencing. Consistent with invadopodia defects in vitro, Endo II silencing led to a significant reduction in tumor growth and metastases in mouse orthotopic tumor xenografts assays performed with both basal breast cancer cell models. This study provides novel insights into roles of Endo II as a positive regulator of invadopodia and basal breast cancer metastasis. Citation Format: Tomas Baldassarre, Jalna Meens, Andrew W. Craig. Endophilin II promotes breast tumor progression and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2013-3796

Abstract 4936: Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop.

Abstract Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src transformed Abl-/-/Arg-/- fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with therapeutic inhibitors of the EGFR invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating an EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib. Citation Format: Karen E. Hayes, Elyse L. Walk, Amanda G. Ammer, Laura C. Kelley, Karen H. Martin, Scott A. Weed. Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4936. doi:10.1158/1538-7445.AM2013-4936

Disparate Species-Dependent Leukocyte Behavior before and after Adding an Elastase Inhibitor

Animal models are often used to identify aspects of the pathophysiology and to develop new therapeutic interventions for human disorders. Leukocyte serine elastases are key mediators of inflammation and meaningful therapeutic targets because of their potential contributions to the Acute Respiratory Distress Syndrome (ARDS) and other inflammatory disorders. We compared the extracellular matrix degrading and bactericidal activities against both Escherichia coli and Staphylococcus aureus, of leukocytes from humans, dogs, hamsters, rats, pigs and rabbits before and after adding highly selective serine elastase inhibitors. Not surprisingly, we found significant species-dependent differences in both activities and the responses to adding serine elastase inhibitors. Our results reinforce the need for care in selecting the optimal animal species for evaluating both the underlying pathophysiology of inflammatory diseases and potential new interventions being developed to treat these conditions.

Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop.

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting that Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src-transformed Abl(-/-)/Arg(-/-) fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib-treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR-invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively, our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.

Functional invadopodia formation through stabilization of the PDPN transcript by IMP-3 and cancer-stromal crosstalk for PDPN expression

We previously reported that insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) depletion (IMP-3(Δ)) was shown to inhibit invadopodia formation and extracellular matrix degradation capacity in oral squamous cell carcinoma (OSCC) cells. In this study, we found that IMP-3(Δ) cells significantly downregulated the podoplanin (PDPN) level, which resulted in a loss of extracellular matrix degradation activity, although invadopodia was still thriving. From RNA in situ hybridization using a digoxigenin-labeled 3'UTR recognition probe of PDPN and reporter assay with 3'UTR of the PDPN gene cloned downstream from the luciferase reporter gene, we revealed that IMP-3 depletion was shown to be downregulated, which most probably lowered PDPN gene expression by reducing mRNA stabilization. In a xenograft model, PDPN depletion was the cause of a decrease in tumor volume and regional infiltration into nearby stroma. Taken together, transforming growth factor beta 1 increased PDPN expression, which potentiated cancer invasion through increased invadopodia formation and extracellular matrix degradation in the low invasive OSCC cell line. Reciprocally, interleukin-1 beta secreted by OSCC cells, stimulated transforming growth factor beta 1 secretion from stromal fibroblasts to induce PDPN expression in OSCC cells. In addition, a retrospective investigation of OSCC patients found that IMP-3 and PDPN expression significantly correlated with lymph node metastasis of OSCC patients. Moreover, co-expression of IMP-3 and PDPN were frequently detected both in primary and lymph nodes metastatic OSCC cells using immunohistochemical dual staining. Thus, the IMP-3-PDPN axis may be a sensitive target molecule in anti-invadopodia therapy for the treatment of metastatic cancers.

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

Role of insulin‐like growth factor‐II mRNA‐binding protein‐3 in invadopodia formation and the growth of oral squamous cell carcinoma in athymic nude mice

The invadopodia are specialized structures that degrade the extracellular matrix (ECM) and promote cell invasion and metastasis. Understanding the forms and functions of invadopodia should facilitate the proper identification of novel targets for antiinvasive therapy. To understand the role of insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) in invadopodia formation and cancer invasion, we performed IMP-3 gene silencing, invadopodia formation, ECM degradation assay, zymography, western blot, and mouse xenograft. We demonstrate that invadopodia evidenced ECM degradation activity in oral squamous cell carcinoma (OSCC). Downregulation of IMP-3 inhibited invadopodia formation, ECM degradation, and tumor growth and invasiveness. Epidermal growth factor receptor (EGFR) signaling may perform a critical function in invadopodia formation, ECM degradation, IMP-3, and cortactin expression. IMP-3 may be intimately correlated with cancer invasion through invadopodia in oral cancer. The overexpression of IMP-3 in oral cancer was predictive of a high correlation with cancer growth and invasion.

The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.

Invadopodia formation in oral squamous cell carcinoma: The role of epidermal growth factor receptor signalling

ObjectiveInvadopodia are actin-rich structures that are formed on the ventral membrane of the cell and degrade extracellular matrix (ECM) by accumulation of matrix metalloproteinase (MMP). Consequently, understanding how invadopodia form and function should facilitate the identification of new therapeutic target for anti-invadopodia therapy. The present study was designed to investigate invadopodia formation associated with oral squamous cell carcinoma (OSCC) and the effect of epidermal growth factor receptor (EGFR) signalling on invadopodia formation and ECM degradation activity. DesignImmunofluorescence analysis of invadopodia formation and ECM degradation was performed using confocal microscope. To understand the role of EGFR signalling, cells were treated with AG1478 or PD153035 (EGF receptor tyrosine kinase inhibitors) and assessed using zymography and an ECM degradation assay. ResultsInvadopodia containing dot-shaped F-actin were observed in stress fibres of HSC-3 OSCC along with evidence of ECM degradation activity. GM6001, a broad range of MMP inhibitor impaired matrix degradation and gelatinolytic activity of active MMP-2. AG1478 and PD153035 inhibited invadopodia formation and ECM degradation activity, as well as gelatinolytic activity of proMMP-9 and proMMP-2. ConclusionsWe provide evidence that HSC-3 OSCC has a tendency to adopt invadopodia for invasion and accompanying MMP-dependent proteolytic ECM degradation and EGFR signalling is necessary for invadopodia formation and associated ECM degradation activity.

Trailblazing LIM kinases take the lead in collective tumor cell invasion

The spread of tumor cells from primary sites often occurs as associated cell collectives. In this form of invasion, the contribution of cells leading the way may differ from those that follow. By implication, proteins that regulate the actin cytoskeleton, a major driver of cell motility, may have different roles depending on whether they are in leading or following cells. The LIM kinases 1 and 2 (LIMK) phosphorylate and inactivate the filamentous actin severing function of cofilin proteins. Using siRNA or pharmacological inhibitors, LIMK was found to be required in leading cells of collectively invading tumor cells, or in cancer-associated stromal fibroblasts, for effective extracellular matrix degradation that facilitates three-dimensional invasion. The decreased extracellular matrix degrading activities were associated with an inability to form the stable filamentous actin structures necessary to make matrix-degrading protrusive structures. However, LIMK was not required for cell motility or for path-following in associated collectives. These findings show that leading and following cells in collective invasion have different properties and indicate that targeting the activities in leading cells is sufficient to significantly inhibit tumor cell invasiveness.

β1A Integrin Is a Master Regulator of Invadosome Organization and Function

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization-depolymerization associated with β1 and β3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using β1⁻/⁻ or β3⁻/⁻ cells, it seemed that β1A but not β3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the β1A tail. Moreover, our study clearly showed that β1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the β1A cytoplasmic domain allowed specific and immediate loss of function of β1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.

Phosphatidylinositol 4,5‐bisphosphate and PIP5‐kinase Iα are required for invadopodia formation in human breast cancer cells

Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and PI(4,5)P(2)-producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P(2) accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P(2) antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P(2) by overexpression of the phospholipase C (PLC) delta1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P(2), also blocked invadopodia formation, while a mutated PLCdelta1-PH domain that lacks PI(4,5)P(2)-binding activity had no effect. Cellular PI(4,5)P(2) production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KIalpha, Ibeta, and Igamma. Real-time quantitative PCR analysis showed that PIP5KIalpha is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIalpha by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIalpha protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIalpha protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P(2) by PIP5KIalpha is required for invadopodia formation and ECM degradation by human breast cancer cells.