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Invadosome is an umbrella term used to describe a family of cellular structures including podosomes and invadopodia. They serve as contact zones between the cell plasma membrane and extracellular matrix, contributing to matrix remodeling by locally enriched proteolytic enzymes. Invadosomes, which are actin-dependent, are implicated in cellular processes promoting adhesion, migration, and invasion. Invadosomes, which exist in various cell types, play crucial roles in physiological phenomena such as vascularization and bone resorption. Invadosomes are also implicated in pathological processes such as matrix tissue remodeling during metastatic tumor cell invasion. This review summarizes basic information and recent advances about mechanisms underlying podosome and invadopodia formation, their organization and function., (© 2024 médecine/sciences – Inserm.)

During cancer progression, tumor cells need to disseminate by remodeling the extracellular tumor matrix. A recent study sheds light on the intricate cooperation between caveolae and invadosomes that facilitates the spread of cancer cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work., (© 2024. The Author(s), under exclusive licence to Society for Mathematical Biology.)

Invadopodia are protrusive structures that mediate the extracellular matrix (ECM) degradation required for tumor invasion and metastasis. Rho small GTPases regulate invadopodia formation, but the molecular mechanisms of how Rho small GTPase activities are regulated at the invadopodia remain unclear. Here we have identified FilGAP, a GTPase-activating protein (GAP) for Rac1, as a negative regulator of invadopodia formation in tumor cells. Depletion of FilGAP in breast cancer cells increased ECM degradation and conversely, overexpression of FilGAP decreased it. FilGAP depletion promoted the formation of invadopodia with ECM degradation. In addition, FilGAP depletion and Rac1 overexpression increased the emergence of invadopodia induced by epidermal growth factor, whereas FilGAP overexpression suppressed it. Overexpression of GAP-deficient FilGAP mutant enhanced invadopodia emergence as well as FilGAP depletion. The pleckstrin-homology (PH) domain of FilGAP binds phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], which is distributed on membranes of the invadopodia. FilGAP localized to invadopodia in breast cancer cells on the ECM, but FilGAP mutant lacking PI(3,4)P 2 -binding showed low localization. Similarly, the decrease of PI(3,4)P 2 production reduced the FilGAP localization. Our results suggest that FilGAP localizes to invadopodia through its PH domain binding to PI(3,4)P 2 and down-regulates invadopodia formation by inactivating Rac1, inhibiting ECM degradation in invasive tumor cells.Key words: invadopodia, breast carcinoma, Rac1, FilGAP, PI(3,4)P 2 .

In a previous study, our research group observed that estrogen promotes the metastasis of non-small cell lung cancer (NSCLC) through the estrogen receptor β (ERβ). Invadopodia are key structures involved in tumor metastasis. However, it is unclear whether ERβ is involved in the promotion of NSCLC metastasis through invadopodia. In our study, we used scanning electron microscopy to observe the formation of invadopodia following the overexpression of ERβ and treatment with E2. In vitro experiments using multiple NSCLC cell lines demonstrated that ERβ can increase the formation of invadopodia and cell invasion. Mechanistic studies revealed that ERβ can upregulate the expression of ICAM1 by directly binding to estrogen-responsive elements (EREs) located on the ICAM1 promoter, which in turn can enhance the phosphorylation of Src/cortactin. We also confirmed these findings in vivo using an orthotopic lung transplantation mouse model, which validated the results obtained from the in vitro experiments. Finally, we examined the expressions of ERβ and ICAM1 using immunohistochemistry in both NSCLC tissue and paired metastatic lymph nodes. The results confirmed that ERβ promotes the formation of invadopodia in NSCLC cells through the ICAM1/p-Src/p-Cortactin signaling pathway., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Purpose: The therapeutic efficacy of radiotherapy/temozolomide treatment for glioblastoma (GBM) is limited by the augmented invasiveness mediated by invadopodia activity of surviving GBM cells. As yet, however the underlying mechanisms remain poorly understood. Due to their ability to transport oncogenic material between cells, small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression. We hypothesize that the sustained growth and invasion of cancer cells depends on bidirectional sEV-mediated cell-cell communication., Methods: Invadopodia assays and zymography gels were used to examine the invadopodia activity capacity of GBM cells. Differential ultracentrifugation was utilized to isolate sEVs from conditioned medium and proteomic analyses were conducted on both GBM cell lines and their sEVs to determine the cargo present within the sEVs. In addition, the impact of radiotherapy and temozolomide treatment of GBM cells was studied., Results: We found that GBM cells form active invadopodia and secrete sEVs containing the matrix metalloproteinase MMP-2. Subsequent proteomic studies revealed the presence of an invadopodia-related protein sEV cargo and that sEVs from highly invadopodia active GBM cells (LN229) increase invadopodia activity in sEV recipient GBM cells. We also found that GBM cells displayed increases in invadopodia activity and sEV secretion post radiation/temozolomide treatment. Together, these data reveal a relationship between invadopodia and sEV composition/secretion/uptake in promoting the invasiveness of GBM cells., Conclusions: Our data indicate that sEVs secreted by GBM cells can facilitate tumour invasion by promoting invadopodia activity in recipient cells, which may be enhanced by treatment with radio-chemotherapy. The transfer of pro-invasive cargos may yield important insights into the functional capacity of sEVs in invadopodia., (© 2023. The Author(s).)

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients' outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease., (© 2022. The Author(s).)

Background: Many studies have shown that diabetes is often closely related to oral squamous cell carcinoma (OSCC) occurrence and metastasis. Heat shock protein 70 (Hsp70) is a molecular chaperone related to diabetes complications. This study aims to investigate the role of Hsp70 in OSCC in expression of invadopodia-associated proteins., Methods: The expressions and correlation of HSP70, Hif1α, MMP2, MMP14, and cortactin were examined using bioinformatics analysis and verified by OSCC tissue microarrays. Assay in vitro was performed to analyze cell migration capacity after treatment with or without the HSP70 inhibitor., Results: The expressions of invadopodia-associated proteins were enhanced in OSCC tissues compared with paracarcinoma tissues and partially correlated with HSP70. Inhibiting HSP70 significantly decreased the cell viability, proliferation, and migration of OSCC cells., Conclusions: HSP70 may be involved in invadopodia-associated proteins in OSCC cells, which provides a promising method for treatment of OSCC metastasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ding, Zhang, Yang, Wu, Su and Wang.)

Non-small cell lung cancer (NSCLC) is one of leading causes of cancer-related mortality worldwide, which harbors various accumulated genetic and epigenetic abnormalities. Histone methyltransferase SETDB1 is a pivotal epigenetic regulator whose focal amplification and upregulation are commonly detected in NSCLC. However, molecular mechanisms underlying the pro-oncogenic function of SETDB1 remain poorly characterized. Here, we demonstrate that SETDB1 augments the migration and invasion capabilities of NSCLC cells by reinforcing invadopodia formation and mediated ECM degradation. At the molecular level, SETDB1 suppresses the expression of FOXA2, a crucial tumor and metastasis suppressor via coordinated epigenetic mechanisms - SETDB1 not only catalyzes histone H3K9 methylation on FOXA2 genomic locus, but also recruits DNMT3A to regulate DNA methylation on CpG island. Consequently, depletion of Setdb1 in murine lung adenocarcinoma cells completely abolished their full and spontaneous metastatic capabilities in mouse xenograft models. These findings together establish the pro-metastasis activity of SETDB1 in NSCLC and elucidate the underlying cellular and molecular mechanisms., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Frank-Ter Haar syndrome (FTHS), sometimes referred to as Ter Haar syndrome, is a rare hereditary disorder that manifests in skeletal, cardiac, and ocular anomalies, including hypertelorism, glaucoma, prominent eyes, and facial abnormalities. In this study, we performed whole-exome sequencing (WES) to identify the genetic component responsible for the phenotype of the index patient, a male infant born to a consanguineous family from Saudi Arabia. The analysis revealed a homozygous missense variant, c.280C>G, in the SH3PXD2B gene, which cosegregates with the familial phenotype with a plausible autosomal-recessive mode of inheritance, indicating a potential disease-causing association. The SH3PXD2B gene encodes a TKS4 podosome adaptor protein that regulates the epidermal growth factor signaling pathway. This study validates the critical function of the TKS4 podosome protein by suggesting a common mechanism underlying the pathogenesis of FTHS.

Bladder cancer invasion depends on mammalian target of rapamycin complex 2 (mTORC2) activity, although the downstream mTORC2 effectors that mediate this effect have not been fully defined. One potential downstream effector is the arginine derivative nitric oxide (NO). This study identified a stage-associated increase in the expression of the NO-generating enzymes endothelial NO synthase (eNOS) and inducible NOS (iNOS) in human bladder cancer. Reduction of NOS activity by pharmacologic inhibition or silencing of NOS enzymes reduced cancer cell invasion, with similar effects observed using the NO scavenger cobinamide. By contrast, enhanced invasion was seen with the NO donor Deta-NONOate and an analog of the downstream NO second messenger cGMP. Next, NOS expression was evaluated in invadopodia, which are cellular protrusions that form the invasive tips of cancer cells. Invadopodia were enriched in both iNOS protein and mTORC2 activity, and invadopodia formation was increased by Deta-NONOate and decreased by cobinamide and ablation of mTORC2 activity. Additionally, mTORC2 increased expression of iNOS. Using a zebrafish model, injection of iNOS- or rictor-silenced cells reduced the frequency of bladder cancer cell metastasis in zebrafish. These results indicate that mTORC2 can mediate bladder cancer cell invasion through increased iNOS expression, resulting in increased NO and cGMP production in invadopodia and further propagation of invadopodia formation., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

At present, there are still many poorly understood aspects of the mechanisms underlying hepatocellular carcinoma (HCC) invasion and metastasis. Invadopodia are important structures for cancer cell invasion and metastasis. We determined that high T-lymphoma invasion and metastasis 1 (Tiam1) expression is associated with HCC invasion and metastasis and poor patient prognosis after surgery. Gain- and loss-of-function studies confirmed that Tiam1 promotes invadopodia formation in HCC by activating Rac1. A series of biochemical experiments confirmed that this effect is regulated by the PI3K/Akt signaling pathway. We also confirmed that PIP2 facilitates this effect. In summary, these findings reveal that Tiam1 plays an important role in invadopodia formation in HCC., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: The BRAF inhibitor, vemurafenib, has been widely used in the treatment of patients with melanoma-bearing BRAF V600E mutations. While the initial response to vemurafenib is usually excellent, the majority of patients eventually develop resistance and metastatic disease. However, the underlying molecular mechanism remains elusive. The objective of this study was therefore to identify additional molecular targets responsible for vemurafenib resistance., Methods: Western blots and immunohistochemistry analyses were used to evaluate expressions of PYK2 and p-PYK2 in cultured cells and melanoma tissue microarrays. The relationships of p-PYK2 with clinicopathological parameters were statistically analyzed. Invadopodia cell invasion, and a Ca 2+ assay were used to determine the effect of vemurafenib resistance-induced p-PYK2 on melanoma progression. A mouse model was used to assess the effects of PYK2 on melanoma metastasis., Results: Elevated p-PYK2 levels were detected in vemurafenib-resistant melanoma cells, and PYK2 was shown to regulate invadopodia formation in melanoma cells. Vemurafenib triggered invadopodia formation by activation of PYK2. Inhibition of PYK2 with either shRNA or the small molecule inhibitor, PF562711, dramatically reduced vemurafenib-induced invadopodia formation. Furthermore, knockdown of PYK2 significantly reduced melanoma lung metastasis in vivo . Increased expressions of p-PYK2 in melanoma patients were positively correlated with advanced stage ( P = 0.002), metastasis ( P < 0.001), and Clark grade ( P < 0.001), and were also associated with short overall survival [hazard ratio (HR) = 3.304, P = 0.007] and progression-free survival (HR = 2.930, P = 0.001)., Conclusions: PYK2 mediated vemurafenib-induced melanoma cell migration and invasion. Inhibition of PYK2 resensitized melanoma cells to vemurafenib. Phospho-PYK2 was a prognostic biomarker in melanoma patients., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Cancer Biology & Medicine.)

Although luminal breast cancer cells are typically highly cohesive epithelial cells and have low invasive ability, many eventually develop metastasis. Until now, the underlying mechanisms remain obscure. In this work, we showed that the level of hyaluronic acid synthase 2 (HAS2) was positively correlated with the malignant phenotype of breast cancer cells. Notably, the increased expression of HAS2 promoted the invasive and migratory abilities of luminal breast cancer cells in vitro, followed by a reduced expression of E-cadherin, β-catenin, and ZO-1, and an elevated expression of N-cadherin and vimentin. Furthermore, overexpression of HAS2 promoted while knockdown of HAS2 impeded invadopodia formation, which subsequently increased or decreased the activation of cortactin, Tks5, and metalloproteinases (MMPs). Activation of these invadopodia-related proteins was prevented by inhibition of HAS2 or disruption of HA, which in turn attenuated the increased motility and invasiveness. Further, in vivo study showed that, HAS2 increased tumor growth and the rate of lung metastasis via driving transition to an invasive cell phenotype in SCID mice that were orthotopically transplanted with luminal breast cancer cells. Collectively, our results showed that HAS2 promoted cell invasion by inducing transition to an invasive phenotype and by enhancing invadopodia formation in luminal breast cancer cells, which may provide new mechanistic insights into its role in tumor metastasis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin's BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN's influence on parameters associated with cells' ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin's BM.

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

Metastasis is the main cause of death in breast cancer patients. The initial step of metastasis is invadopodia-mediated extracellular matrix (ECM) degradation, which enables local breast tumor cells to invade surrounding tissues. However, the molecular mechanism underlying invadopodia-mediated metastasis remains largely unknown. Here we found that the RNA-binding protein Musashi1 (Msi1) exhibited elevated expression in invasive breast tumors and promoted lung metastasis of mammary cancer cells. Suppression of Msi1 reduced invadopodia formation in mammary cancer cells. Furthermore, Msi1 deficiency decreased the expression and activity of Mmp9, an important enzyme in ECM degradation. Mechanistically, Msi1 directly suppressed Timp3, an endogenous inhibitor of Mmp9. In clinical breast cancer specimens, TIMP3 and MSI1 levels were significantly inversely correlated both in normal breast tissue and breast cancer tissues and associated with overall survival in breast cancer patients. Taken together, our findings demonstrate that the MSI1-TIMP3-MMP9 cascade is critical for invadopodia-mediated onset of metastasis in breast cancer, providing novel insights into a promising therapeutic strategy for breast cancer metastasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Oral squamous cell carcinoma (OSCC) is the most relevant malignant neoplasm among all head and neck tumours due to its high prevalence and unfavourable prognosis. Tumour invasion and metastasis that affect prognosis are result of a set of complex events that cells with invasive potential use to spread to other regions. These cells use several mechanisms to invade tissues, including a type of finger-like membrane protrusion called invadopodia. This study aims to investigate the immunoexpression of invaopodia related-proteins TKs5, cortactin, TKs4 and MT1-MMP in OSCC and correlate it to clinicopathological data., Methods: An immunohistochemical evaluation of fifty cases of OSCCs and 20 cases of oral mucosa (OM) were assessed. The expression of invadopodia proteins were analysed in comparison to normal tissue (OM) and correlated to different clinical-stage and histological grade of OSCC., Results: TKs5, cortactin, TKs4 and MT1-MMP were significantly overexpressed in OSCC when compared to OM (p < 0.0001). Among tumour stages, TKs5 showed a statistical difference in immunolabelling between stage I and III (p = 0.026). Cortactin immunolabelling was statistically higher in grade I than in grade II and III. No differences were seen on TKs4 expression based on tumour staging or grading. MT1-MMP was higher expressed and showed statistical difference between stages I and III and grades I compared to II and III., Conclusions: The invadopodia related-proteins were found to be overexpressed in OSCC when compared to OM, suggesting invadopodia formation and activity. Besides overexpressed in OSCC, cortactin, TKs4 and TKs5 showed no or ambiguous differences in protein expression when compared among clinical-stages or histological grades groups. Conversely, the expression of MT1-MMP increased in advanced stages and less differentiated tumours, suggesting MT1-MMP expression as a promising prognostic marker in OSCC.

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum +/+ and Lum -/- mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum -/- mice as compared to Lum +/+ , suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/β. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo . A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target., (©2021 American Association for Cancer Research.)

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ET A R) drives invadopodia by a not fully explored coordinated function of β-arrestin1 (β-arr1). Here, we report that β-arr1 links the integrin-linked kinase (ILK)/βPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ET A R/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ET A R antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/β-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that β1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between β1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates β1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with β1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of β1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates β1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity and is usually preceded by a range of premalignant tissue abnormalities termed oral potentially malignant disorders. Identifying malignant transformation is critical for early treatment and consequently improved survival and decreased morbidity. Invadopodia (INV) are specialized subcellular structures required for cancer cell invasion. We developed a new method to visualize INV in keratinocytes using fluorescent immunohistochemistry (FIHC) and semi-automated images analysis. The presence of INV was used to determine the risk of malignant transformation. We analyzed 145 formalin-fixed, paraffin-embedded (FFPE) oral biopsy samples from 95 patients diagnosed as nondysplastic, dysplastic, and OSCC including 49 patients whose lesions transformed to OSCC (progressing) and 46 cases that did not transform to OSCC (control). All samples were stained for Cortactin, tyrosine kinase substrate with five SH3 domains (Tks5) and matrix metallopeptidase 14 (MMP14) using FIHC, imaged using confocal microscopy and analyzed using a multichannel colocalization analysis. The areas of colocalization were used to generate an INV score. Using the INV score, we were able to identify progressing lesions with a sensitivity of 75-100% and specificity of 72-76%. A positive INV score was associated with increased risk of progression to OSCC. Our results suggest that INV markers can be used in conjunction with the current diagnostic standard for early detection of OSCC., (© 2020 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd.)

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.

Background: Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549., Methods: We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation., Results: We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation., Conclusion: In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. Video abstract.

Hypoxia plays a significant role in solid tumors by the increased expression of hypoxia-inducible factor-1α (HIF-1α), which is known to promote cancer invasion and metastasis. Cancer-cell invasion dynamically begins with the degradation of the extracellular matrix (ECM) via invadopodia formation. The chemical substrates that are utilized by hypoxic cells as fuel to drive invadopodia formation are still not fully understood. Therefore, the aim of the study was to maintain MDA-MB-231 cells under hypoxia conditions to allow cells to form a large number of invadopodia as a model, followed by identifying their nutrient utilization. The results of the study revealed an increase in the number of cells forming invadopodia under hypoxia conditions. Moreover, Western blot analysis confirmed that essential proteins for hypoxia and invadopodia, including HIF-1α, vascular endothelial growth factor (VEGF), metallopeptidase-2 (MMP-2), and Rho guanine nucleotide exchange factor 7 (β-PIX), significantly increased under hypoxia. Interestingly, phenotype microarray showed that only 11 chemical substrates from 367 types of substrates were significantly metabolized in hypoxia compared to in normoxia. This is thought to be fuel for hypoxia to drive the invasion process. In conclusion, we found 11 chemical substrates that could have potential energy sources for hypoxia-induced invadopodia formation of these cells. This may in part be a target in the hypoxic tumor and invadopodia formation. Additionally, these findings can be used as potential carrier targets in cancer-drug discovery, such as the usage of dextrin.

Cancer cells break tissue barriers by use of small actin-rich membrane protrusions called invadopodia. Complete invadopodia maturation depends on protrusion outgrowth and the targeted delivery of the matrix metalloproteinase MT1-MMP via endosomal transport by mechanisms that are not known. Here, we show that the ER protein Protrudin orchestrates invadopodia maturation and function. Protrudin formed contact sites with MT1-MMP-positive endosomes that contained the RAB7-binding Kinesin-1 adaptor FYCO1, and depletion of RAB7, FYCO1, or Protrudin inhibited MT1-MMP-dependent extracellular matrix degradation and cancer cell invasion by preventing anterograde translocation and exocytosis of MT1-MMP. Moreover, when endosome translocation or exocytosis was inhibited by depletion of Protrudin or Synaptotagmin VII, respectively, invadopodia were unable to expand and elongate. Conversely, when Protrudin was overexpressed, noncancerous cells developed prominent invadopodia-like protrusions and showed increased matrix degradation and invasion. Thus, Protrudin-mediated ER-endosome contact sites promote cell invasion by facilitating translocation of MT1-MMP-laden endosomes to the plasma membrane, enabling both invadopodia outgrowth and MT1-MMP exocytosis., (© 2020 Pedersen et al.)

Liver metastases remain a major cause of death from gastrointestinal tract cancers and other malignancies, such as breast and lung carcinomas. Understanding the underlying biology is essential for the design of effective therapies. We previously identified the chemokine CCL7 and its receptor CCR3 as critical mediators of invasion and metastasis in lung and colon carcinoma cells. Here we show that the CCL7/CCR3 axis regulates a late stage in invadopodia genesis namely, the targeting of MMP-9 to the invadopodia complex, thereby promoting invadopodia maturation and collagen degradation. We show that this process could be blocked by overexpression of a dominant negative RhoA in highly invasive cells, while a constitutively active RhoA upregulated invadopodia maturation in CCL7-silenced and poorly invasive and metastatic cells and also enhanced their metastatic potential in vivo, collectively, implicating RhoA activation in signaling downstream of CCL7. Blockade of the ERK or PI3K pathways by chemical inhibitors also inhibited invadopodia formation, but affected the initiation stage of invadopodia genesis. Our data implicate CCL7/CCR3 signaling in invadopodia maturation and suggest that chemokine signaling acts in concert with extracellular matrix-initiated signals to promote invasion and liver metastasis., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Overactivation and excessive differentiation of osteoclasts (OCs) has been implicated in the course of bone metabolism-related diseases. Although fullerenol nanoparticles (fNPs) have been suggested to inhibit OC differentiation and OC function in our previous work, systemic studies on the effect of fNPs on bone diseases, e.g., osteoporosis (OP), in vivo remain elusive. Herein, it is demonstrated that fNPs significantly suppress the differentiation of OCs that derived from the murine bone marrow monocytes and inhibit the formation of the sealing zone by blocking the formation and patterning of podosomes in OCs spatiotemporally. In vivo, fNPs are supposed to be an efficient inhibitor of the overactivation of OCs in a LPS-induced bone erosion mouse model. The therapeutic effect of fNPs on osteoporosis is also investigated in an ovariectomy-induced osteoporosis rat model. The well-organized trabecular bone, the reduction in the number of TRAP positive cells, the improvement of bone-associated parameters, and the mechanical properties all demonstrate that fNPs, similar to diphosphonates, can be a promising candidate for the effective treatment of osteoporosis.

CR3 and CR4, the leukocyte specific β2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of β2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: BS and BF are employees of CellSorter Company for Innovations. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Moesin has been proved to be implicated in invasiveness and metastasis in many other cancers, but unclear in HCC. Thus, this study was performed to investigate the clinical significance of moesin and its biological functions in HCC. The results showed that moesin was significantly up-regulated in HCC tissues and was an independent prognostic factor for predicting the recurrence of HCC patients, postoperatively. Furthermore, we also demonstrated that moesin promoted the migration and invasion of HCC cells in vitro and in vivo. And the mechanism studies indicated that moesin overexpression increased the formation of invadopodia and improved the activation of β-catenin/MMP9 axis. Taken together, our findings revealed that moesin acted as an important onco-protein participating in the metastasis of HCC., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanobiology plays a prominent role in cancer invasion and metastasis. The ability of a cancer to degrade extracellular matrix (ECM) is likely connected to its invasiveness. Many cancer cells form invadopodia-micrometer-sized cellular protrusions that promote invasion through matrix degradation (proteolysis). Although it has been hypothesized that invadopodia exert mechanical force that is implicated in cancer invasion, direct measurements remain elusive. Here, we use a recently developed interferometric force imaging technique that provides piconewton resolution to quantify invadopodial forces in cells of head and neck squamous carcinoma and to monitor their temporal dynamics. We compare the force exerted by individual protrusions to their ability to degrade ECM and investigate the mechanical effects of inhibiting invadopodia through overexpression of microRNA-375. By connecting the biophysical and biochemical characteristics of invadopodia, our study provides a new perspective on cancer invasion that, in the future, may help to identify biomechanical targets for cancer therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Osteoclasts are responsible for bone resorption and play an important role in physiological and pathological bone metabolism. Osteoclast migration across bone surfaces is essential for bone resorption, and a previous study demonstrated the role of autophagy in osteoclastogenesis and acid secretion. However, the role of autophagy in osteoclast migration remains unclear. Osteoclast migration requires the successive and rapid assembly and disassembly of podosome rings. In this study, we show that kindlin3, an important adaptor protein in the podosome, can interact with LC3B and undergo autophagy-mediated protein degradation to promote the disassembly of the podosome. Moreover, further analyses showed that the inhibition of autophagy increased kindlin3 levels and enhanced the interaction between kindlin3 and integrin β3. The over activation of integrins inhibits the disassembly of obsolete podosome rings, resulting in disorganization of the actin cytoskeleton and impaired migration in osteoclasts. Our results show that LC3B affects osteoclast migration and FAK/AKT activation by modulating integrin activation via a kindlin3-mediated inside-out signal from the extracellular matrix. Based on these results, we propose that LC3 is an important target for regulating osteoclast migration., (Copyright © 2019. Published by Elsevier Inc.)

Hypoxia-inducible factor-1α (HIF-1α) induces cancer metastasis. We previously demonstrated that HIF-1α-induced membrane-type 4 matrix metalloproteinase (MT4-MMP) is involved in hypoxia-mediated metastasis in head and neck squamous cell carcinoma (HNSCC). However, the functions and detailed mechanisms of MT4-MMP in cancer metastasis are not well understood. In this study, we investigated whether MT4-MMP regulates invadopodia formation or individual cell movement-both critical to cancer migration and invasion-in three-dimensional (3D) environments. By expressing MT4-MMP in the HNSCC cell line FaDu, we demonstrated that MT4-MMP increases invadopodia formation and gelatin degradation. Furthermore, the amoeboid-like cell movement on collagen gel was increased by MT4-MMP expression in FaDu cells. Mechanistically, MT4-MMP may induce invadopodia formation by binding with Tks5 and PDGFRα to result in Src activation and promote amoeboid-like movement by stimulating the small GTPases Rho and Cdc42. Altogether, our data indicate that MT4-MMP induces two crucial mechanisms of cancer dissemination, invadopodia formation and amoeboid movement, and elucidate the prometastatic role of MT4-MMP in hypoxia-mediated cancer metastasis., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: MicroRNA-612 (miR-612) has been proven to suppress EMT, stemness, and tumor metastasis of hepatocellular carcinoma (HCC) via PI3K/AKT2 and Sp1/Nanog signaling. However, its biological roles on HCC progression are far from elucidated., Methods: We found direct downstream target of miR-612, hadha by RNA immunoprecipitation and sequencing. To explore its biological characteristic, potential molecular mechanism, and clinical relevance in HCC patients, we performed several in-vitro and in-vivo models, as well as human tissue chip., Results: Ectopic expression of miR-612 could partially reverse the level of HADHA, then suppress function of pseudopods, and diminish metastatic and invasive potential of HCC by lipid reprogramming. In detail, miR-612 might reduce invadopodia formation via HADHA-mediated cell membrane cholesterol alteration and accompanied with the inhibition of Wnt/β-catenin regulated EMT occurrence. Our results showed that the maximum oxygen consumption rates (OCR) of HCCLM3 miR-612-OE and HCCLM3 hadha-KD cells were decreased nearly by 40% and 60% of their counterparts (p < 0.05). The levels of acetyl CoA were significantly decreased, about 1/3 (p > 0.05) or 1/2 (p < 0.05) of their controls, in exogenous miR-612 or hadha-shRNA transfected HCCLM3 cell lines. Besides, overexpression of hadha cell lines had a high expression level of total cholesterol, especially 27-hydroxycholesterol (p < 0.005). SREBP2 protein expression level as well as its downstream targets, HMGCS1, HMGCR, MVD, SQLE were all deregulated by HADHA. Meanwhile, the ATP levels were reduced to 1/2 and 1/4 in HCCLM3 miR-612-OE (p < 0.05) and HCCLM3 hadha-KD (p < 0.01) respectively. Moreover, patients with low miR-612 levels and high HADHA levels had a poor prognosis with shorter overall survival., Conclusion: miR-612 can suppress the formation of invadopodia, EMT, and HCC metastasis and by HADHA-mediated lipid programming, which may provide a new insight of miR-612 on tumor metastasis and progression.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-β1, using engineered gratings with different width and depth. We first report that TGF-β1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-β1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells., (Copyright © 2019. Published by Elsevier Ltd.)

The basement membrane (BM) provides a physical barrier to invasion in epithelial tumors, and alterations in the molecular makeup and structural integrity of the BM have been implicated in cancer progression. Invadopodia are the invasive protrusions that enable cancer cells to breach the nanoporous basement membrane, through matrix degradation and generation of force. However, the impact of covalent cross-linking on invadopodia extension into the BM remains unclear. Here, we examine the impact of covalent cross-linking of extracellular matrix on invasive protrusions using biomaterials that present ligands relevant to the basement membrane and provide a nanoporous, confining microenvironment. We find that increased covalent cross-linking of reconstituted basement membrane (rBM) matrix diminishes matrix mechanical plasticity, or the ability of the matrix to permanently retain deformation due to force. Covalently cross-linked rBM matrices, and rBM-alginate interpenetrating networks (IPNs) with covalent cross-links and low plasticity, restrict cell spreading and protrusivity. The reduced spreading and reduced protrusivity in response to low mechanical plasticity occurred independent of proteases. Mechanistically, our computational model reveals that the reduction in mechanical plasticity due to covalent cross-linking is sufficient to mechanically prevent cell protrusions from extending, independent of the impact of covalent cross-linking or matrix mechanical plasticity on cell signaling pathways. These findings highlight the biophysical role of covalent cross-linking in regulating basement membrane plasticity, as well as cancer cell invasion of this confining tissue layer., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Monocytes are a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC). However, the complex interactions between tumor cells and monocytes and their role in tumor invasion have not been fully established., Methods: To specifically test the impact of interaction on invasive potential two PDAC cell lines PaTu8902 and CFPAC-1 were selected on their ability to form invasive adhesions, otherwise known as invadopodia and invade in a spheroid invasion assay., Results: Interestingly when the PDAC cells were co-cultured with undifferentiated THP1 monocyte-like cells invadopodia formation was significantly suppressed. Moreover, conditioned media of THP1 cells (CM) was also able to suppress invadopodia formation. Further investigation revealed that both tissue inhibitor of metalloproteinase (TIMP) 1 and 2 were present in the CM. However, suppression of invadopodia formation was found that was specific to TIMP2 activity., Conclusions: Our findings indicate that TIMP2 levels in the tumour microenvironment may have prognostic value in patients with PDAC. Furthermore, activation of TIMP2 expressing monocytes in the primary tumour could present a potential therapeutic opportunity to suppress cell invasion in PDAC.

CDC42 interacting protein 4 (CIP4) is a CDC42 effector that coordinates membrane deformation and actin polymerization. The correlation of CIP4 overexpression with metastatic capacity has been characterized in several types of cancer. However, little information exists on how CIP4 function is regulated. CIP4 interacts with A-kinase (PKA) anchoring protein 350 (AKAP350) and CIP4 is also a PKA substrate. Here, we identified CIP4 T225 as the major CIP4 PKA phosphorylation site. In vitro and in vivo experiments using hepatocellular carcinoma (HCC) and breast cancer cells showed that expression of a CIP4(T225E) phosphomimetic mutant increased cancer cell metastatic capacity and that, conversely, expression of a CIP4(T225A) non-phosphorylatable mutant reduced invasive properties. PKA inhibition decreased to CIP4(T225A) cell-levels control but not CIP4(T225E) cell migratory and invasive efficiency. Concomitantly, our studies indicate that CIP4 T225 phosphorylation promotes the formation of functional invadopodia and enhances CIP4 localization at these structures. Our findings further provide mechanistic data indicating that CIP4 T225 phosphorylation facilitates CIP4 interaction with CDC42. Altogether this study identifies a signaling pathway that involves CIP4 phosphorylation by PKA during the acquisition of a metastatic phenotype in cancer cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Metastatic bladder cancer (BLCA) is a lethal disease with an unmet need for study. Transgelin (TAGLN) is an actin-binding protein that affects the dynamics of the actin cytoskeleton indicating its robust potential as a metastasis initiator. Here, we sought to explore the expression pattern of TAGLN and elucidate its specific functioning and mechanisms in BLCA., Methods: A comprehensive assessment of TAGLN expression in BLCA was performed in three cohorts with a total of 847 patients. The potential effects of TAGLN on BLCA were further determined using clinical genomic analyses that guided the subsequent functional and mechanistic studies. In vitro migration, invasion assays and in vivo metastatic mouse model were performed to explore the biological functions of TAGLN in BLCA cells. Immunofluorescence, western blot and correlation analysis were used to investigate the molecular mechanisms of TAGLN., Findings: TAGLN was highly expressed in BLCA and correlated with advanced prognostic features. TAGLN promoted cell colony formation and cell migration and invasion both in vitro and in vivo by inducing invadopodia formation and epithelial-mesenchymal transition, during which a significant correlation between TAGLN and Slug was observed. The progression-dependent correlation between TGF-β and TAGLN was analysed at both the cellular and tissue levels, while TGF-β-mediated migration was abolished by the suppression of TAGLN., Interpretation: Overall, TAGLN is a promising novel prognosis biomarker of BLCA, and its metastatic mechanisms indicate that TAGLN may represent a novel target agent that can be utilized for the clinical management of invasive and metastatic BLCA. FUND: This work was supported by the National Natural Science Foundation of China [81772703, 81672546, 81602253]; the Natural Science Foundation of Beijing [71772219, 7152146]. and Innovative Fund for Doctoral Students of Peking University Health Science Center (BUM2018BSS002). Funders had no role in the design of the study, data collection, data analysis, interpretation, or the writing of this report., (Copyright © 2019. Published by Elsevier B.V.)

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. For instance, increased arterial stiffness can lead to atherosclerosis, while stiffening of the heart due to fibrosis can increase the chances of heart failure. Cells can sense the stiffness of the extracellular matrix through integrin adhesions and other mechanosensitive structures and in response to this initiate mechanosignalling pathways that ultimately change the cellular behaviour. Over the past decades, interest in mechanobiology has steadily increased and with this also our understanding of the molecular basis of mechanosensing and transduction. However, much of our knowledge about the mechanisms is derived from studies investigating focal adhesions in non-muscle cells, which are distinct in several regards from the cell-matrix adhesions in cardiomyocytes (costameres) or vascular smooth muscle cells (dense plaques or podosomes). Therefore, we will look here first at the evidence for mechanical sensing in the cardiovascular system, before comparing the different cytoskeletal arrangements and adhesion sites in cardiomyocytes and vascular smooth muscle cells and what is known about mechanical sensing through the various structures.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. SIGNIFICANCE: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma., (©2019 American Association for Cancer Research.)

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1-expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process., (© 2018 Qiang et al.)

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbed TM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using Cellbed TM accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Hypoxia, a common feature of solid tumors, has been critically involved in cell invasion and metastasis, but the underlying mechanisms remain poorly understood. Previously, it has been observed that the lysophosphatidic acid receptor 4 (LPA 4 ) signaling axis mediates production of the degradative subcellular structures invadopodia, which are known to be required for metastasis. Here, it is demonstrated that LPA 1 (LPAR1) is a common and major receptor used for hypoxia-induced invadopodia production in various cancer cell lines. The widespread use of LPA 1 was not due to increased LPA 1 expression but rather relied on Src-mediated cross-talk with EGFR. LPA 1 -mediated phosphorylation of Y845-EGFR under hypoxia led to PI3K/Akt activation, an event that increases the ability of cells to produce invadopodia. Moreover, phospho-Y845-EGFR was upregulated in hypoxic zones of tumors and a combination of EGFR and LPA 1 inhibition synergistically suppressed metastasis in vivo Implications: This study uncovers an LPA 1 -EGFR signaling axis that is used for cell invasion in hypoxia and suggests a potential target to impede cancer metastasis. Mol Cancer Res; 16(10); 1601-13. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: The rapid increase in the incidence of head and neck squamous cell carcinoma (HNSCC) is caused by high-risk human papillomavirus (HPV) infections. The HPV oncogenes E6 and E7 promote carcinogenesis by disrupting signaling pathways that control survival and proliferation. Although these cancers are often diagnosed with metastases, the mechanisms that regulate their dissemination are unknown., Aims: The aim of this study was to determine whether the HPV-16 E6 and E7 oncogenes affected the invasive and migratory properties of HNSCC cells which promote their spread and metastasis., Methods and Results: Invasiveness was determined using invadopodia assays which allow for quantitation of extracellular matrix (ECM) degradation by invadopodia which are proteolytic membrane protrusions that facilitate invasion. Using cell lines and genetic manipulations, we found that HPV inhibited invadopodia activity in aggressive cell lines which was mediated by the E6 and E7 oncogenes. Given these findings, we also tested whether HPV caused differences in the migratory ability of HNSCC cells using Transwell assays. In contrast to our invadopodia results, we found no correlation between HPV status and cell migration; however, blocking the expression of the E6 and E7 oncoproteins in a HPV-positive (HPV + ) HNSCC cell line resulted in decreased migration., Conclusions: Our data suggest that the E6 and E7 oncoproteins are negative regulators of invadopodia activity but may promote migration in HPV + HNSCC cells. Despite the need for ECM proteolysis to penetrate most tissues, the unique structure of the head and neck tissues in which these cancers arise may facilitate the spread of migratory cancer cells without significant proteolytic ability., (© 2018 The Authors. Cancer Reports Published by Wiley Periodicals, Inc.)