Understanding the molecular mechanisms of human diseases: the benefits of fission yeasts.

The role of model organisms such as yeasts in life science research is crucial. Although the baker's yeast (Saccharomyces cerevisiae) is the most popular model among yeasts, the contribution of the fission yeasts (Schizosaccharomyces) to life science is also indisputable. Since both types of yeasts share several thousands of common orthologous genes with humans, they provide a simple research platform to investigate many fundamental molecular mechanisms and functions, thereby contributing to the understanding of the background of human diseases. In this review, we would like to highlight the many advantages of fission yeasts over budding yeasts. The usefulness of fission yeasts in virus research is shown as an example, presenting the most important research results related to the Human Immunodeficiency Virus Type 1 (HIV-1) Vpr protein. Besides, the potential role of fission yeasts in the study of prion biology is also discussed. Furthermore, we are keen to promote the uprising model yeast Schizosaccharomyces japonicus, which is a dimorphic species in the fission yeast genus. We propose the hyphal growth of S. japonicus as an unusual opportunity as a model to study the invadopodia of human cancer cells since the two seemingly different cell types can be compared along fundamental features. Here we also collect the latest laboratory protocols and bioinformatics tools for the fission yeasts to highlight the many possibilities available to the research community. In addition, we present several limiting factors that everyone should be aware of when working with yeast models.

Ubiquitin-Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma.

Tumor-derived extracellular vesicles (EVs) are potential biomarkers for tumors, but their reliable molecular targets have not been identified. The previous study confirms that ubiquitin-specific protease 22 (USP22) promotes lung adenocarcinoma (LUAD) metastasis in vivo and in vitro. Moreover, USP22 regulates endocytosis of tumor cells and localizes to late endosomes. However, the role of USP22 in the secretion of tumor cell-derived EVs remains unknown. In this study, it demonstrates that USP22 increases the secretion of tumor cell-derived EVs and accelerates their migration and invasion, invadopodia formation, and angiogenesis via EV transfer. USP22 enhances EV secretion by upregulating myosin IB (MYO1B). This study further discovers that USP22 activated the SRC signaling pathway by upregulating the molecule KDEL endoplasmic reticulum protein retention receptor 1 (KDELR1), thereby contributing to LUAD cell progression. The study provides novel insights into the role of USP22 in EV secretion and cell motility regulation in LUAD.

Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Vesicle transport of matrix metalloproteinases.

Multicellular organisms consist of cells and extracellular matrix (ECM). ECM creates a cellular microenvironment, and cells locally degrade the ECM according to their cellular activity. A major group of enzymes that modify ECM belongs to matrix metalloproteinases (MMPs) and play major roles in various pathophysiological events. ECM degradation by MMPs does not occur in all cellular surroundings but only where it is necessary, and cells achieve this by directionally secreting these proteolytic enzymes. Recent studies have indicated that such enzyme secretion is achieved by targeted vesicle transport along the microtubules, and several kinesin superfamily proteins (KIFs) have been identified as responsible motor proteins involved in the processes. This chapter discusses recent findings of the vesicle transport of MMPs and their roles.

PAK6 acts downstream of IQGAP3 to promote contractility in triple negative breast cancer cells.

Breast cancer is a heterogeneous disease that remains the most common malignancy among women worldwide. During genomic analysis of breast tumours, mRNA levels of IQGAP3 were found to be upregulated in triple negative tumours. IQGAP3 was subsequently found to be expressed across a panel of triple negative breast cancer (TNBC) cell lines. Depleting expression levels of IQGAP3 delivered elongated cells, disrupted cell migration, and inhibited the ability of cells to form specialised invasive adhesion structures, termed invadopodia. The morphological changes induced by IQGAP3 depletion were found to be dependent on RhoA. Indeed, reduced expression of IQGAP3 disrupted RhoA activity and actomyosin contractility. Interestingly, IQGAP3 was also found to interact with p-21 activated kinase 6 (PAK6); a protein already associated with the regulation of cell morphology. Moreover, PAK6 depletion phenocopied IQGAP3 depletion in these cells. Whereas PAK6 overexpression rescued the IQGAP3 depletion phenotype. Our work points to an important PAK6-IQGAP3-RhoA pathway that drives the cellular contractility of breast cancer cells promoting both cell migration and adhesive invasion of these cells. As this phenotype is relevant to the process of metastasis and re-seeding of metastasis, the pharmacological targeting of PAK6 could lead to clinical benefit in TNBC patients.

Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. METHODS: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. RESULTS: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. CONCLUSIONS: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Nuclear transport of phosphorylated LanCL2 promotes invadopodia formation and tumor progression of glioblastoma by activating STAT3/Cortactin signaling.

INTRODUCTION: Our previous study showed that the abscisic acid receptor lanthionine synthetase C-like 2 (LanCL2) is a significant prognostic factor for overall survival in young glioblastoma patients. However, the role of LanCL2 in glioblastoma remains unclear yet. OBJECTIVES: This study aims to investigate the role of LanCL2 in regulating in-vitro cell invasion and in-vivo tumor progression of glioblastoma and its underlying mechanism. METHODS: Tyrosine 198 or 295 residue of LanCL2 was mutated using site-directed mutagenesis to block its phosphorylation. The role of LanCL2 in glioblastoma was investigated using transwell or 3D invasion assay, matrix degradation assay and intracranial xenograft model. RESULTS: This study showed that nuclear transport of LanCL2 was enhanced by overexpression of LanCL2 or its ligand abscisic acid in glioblastoma cells. Knockdown of LanCL2 suppressed migration, invasion and invadopodia formation of glioblastoma cells, whereas overexpression of wild-type LanCL2 enhanced them. Blocking of Tyr295 residue phosphorylation of LanCL2 impeded its nuclear transport, retarded glioblastoma cell motility and invadopodia formation, and deceased the phosphorylation of Cortactin and STAT3. c-Met was identified as the upstream tyrosine kinase of Tyr295 residue of LanCL2, and inhibition of c-Met markedly suppressed the nuclear transport of LanCL2. Moreover, overexpression of wild-type LanCL2 significantly promoted orthotopic tumor growth of glioblastoma in vivo and led to poor survival of mice with median survival time of 33.5 days, whereas Tyr295 mutation rescued it with median survival time of 49 days. CONCLUSION: Our findings suggested that Tyr295 phosphorylation is crucial to the activation and nuclear transport of LanCL2, as well as invadopodia formation and tumor progression of glioblastoma, providing the evidence of a novel signaling axis c-Met/LanCL2/STAT3/Cortactin and the first observation of the importance of Tyr295 phosphorylation to LanCL2.

KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cross-talk between gastric cancer and hepatic stellate cells promotes invadopodia formation during liver metastasis.

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRß) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM.

A synergistic regulation works in matrix stiffness-driven invadopodia formation in HCC.

Growing evidence has suggested that increased matrix stiffness can significantly strengthen the malignant characteristics of hepatocellular carcinoma (HCC) cells. However, whether and how increased matrix stiffness regulates the formation of invadopodia in HCC cells remain largely unknown. In the study, we developed different experimental systems in vitro and in vivo to explore the effects of matrix stiffness on the formation of invadopodia and its relevant molecular mechanism. Our results demonstrated that increased matrix stiffness remarkably augmented the migration and invasion abilities of HCC cells, upregulated the expressions of invadopodia-associated genes and enhanced the number of invadopodia. Two regulatory pathways contribute to matrix stiffness-driven invadopodia formation together in HCC cells, including direct triggering invadopodia formation through activating integrin ß1 or Piezo1/ FAK/Src/Arg/cortactin pathway, and indirect stimulating invadopodia formation through improving EGF production to activate EGFR/Src/Arg/cortactin pathway. Src was identified as the common hub molecule of two synergistic regulatory pathways. Simultaneously, activation of integrin ß1/RhoA/ROCK1/MLC2 and Piezo1/Ca2+/MLCK/MLC2 pathways mediate matrix stiffness-reinforced cell migration. This study uncovers a new mechanism by which mechanosensory pathway and biochemical signal pathway synergistically regulate the formation of invadopodia in HCC cells.

Fibroblast activation protein drives tumor metastasis via a protease-independent role in invadopodia stabilization.

During metastasis, tumor cells invade through the basement membrane and intravasate into blood vessels and then extravasate into distant organs to establish metastases. Here, we report a critical role of a transmembrane serine protease fibroblast activation protein (FAP) in tumor metastasis. Expression of FAP and TWIST1, a metastasis driver, is significantly correlated in several types of human carcinomas, and FAP is required for TWIST1-induced breast cancer metastasis to the lung. Mechanistically, FAP is localized at invadopodia and required for invadopodia-mediated extracellular matrix degradation independent of its proteolytic activity. Live cell imaging shows that association of invadopodia precursors with FAP at the cell membrane promotes the stabilization and growth of invadopodia precursors into mature invadopodia. Together, our study identified FAP as a functional target of TWIST1 in driving tumor metastasis via promoting invadopodia-mediated matrix degradation and uncovered a proteolytic activity-independent role of FAP in stabilizing invadopodia precursors for maturation.

Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics.

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

Invadopodia associated Thrombospondin-1 contributes to a post-therapy pro-invasive response in glioblastoma cells.

A critical challenge in the treatment of glioblastoma (GBM) is its highly invasive nature which promotes cell migration throughout the brain and hinders surgical resection and effective drug delivery. GBM cells demonstrate augmented invasive capabilities following exposure to the current gold standard treatment of radiotherapy (RT) and concomitant and adjuvant temozolomide (TMZ), resulting in rapid disease recurrence. Elucidating the mechanisms employed by post-treatment invasive GBM cells is critical to the development of more effective therapies. In this study, we utilized a Nanostring® Cancer Progression gene expression panel to identify candidate genes that may be involved in enhanced GBM cell invasion after treatment with clinically relevant doses of RT/TMZ. Our findings identified thrombospondin-1 (THBS1) as a pro-invasive gene that is upregulated in these cells. Immunofluorescence staining revealed that THBS1 localised within functional matrix-degrading invadopodia that formed on the surface of GBM cells. Furthermore, overexpression of THBS1 resulted in enhanced GBM cell migration and secretion of MMP-2, which was reduced with silencing of THBS1. The preliminary data demonstrates that THBS1 is associated with invadopodia in GBM cells and is likely involved in the invadopodia-mediated invasive process in GBM cells exposed to RT/TMZ treatment. Therapeutic inhibition of THBS1-mediated invadopodia activity, which facilitates GBM cell invasion, should be further investigated as a treatment for GBM.

Analysis of Genes Related to Invadopodia Formation and CTTN in Oral Squamous Cell Carcinoma-A Systematic Gene Expression Analysis.

Successful treatment for any type of carcinoma largely depends on understanding the patterns of invasion and migration. For oral squamous cell carcinoma (OSCC), these processes are not entirely understood as of now. Invadopodia and podosomes, called invadosomes, play an important role in cancer cell invasion and migration. Previous research has established that cortactin (CTTN) is a major inducer of invadosome formation. However, less is known about the expression patterns of CTTN and other genes related to it or invadopodia formation in OSCC during tumor progression in particular. In this study, gene expression patterns of CTTN and various genes (n = 36) associated with invadopodia formation were analyzed to reveal relevant expression patterns and give a comprehensive overview of them. The genes were analyzed from a whole genome dataset of 83 OSCC samples relating to tumor size, grading, lymph node status, and UICC (Union for Internatioanl Cancer Control). The data revealed significant overexpression of 18 genes, most notably CTTN, SRC (SRC proto-onocogene, non-receptor tyrosine kinase), EGFR (epidermal growth factor receptor), SYK (spleen associated tyrosine kinase), WASL (WASP like actin nucleation promotion factor), and ARPC2 (arrestin beta 1) due to their significant correlation with further tumor parameters. This study is one of the first to summarize the expression patterns of CTTN and related genes in a complex group of OSCC samples.

Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and the establishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery.

FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells.

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)P2], 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)P2-binding showed low localization. Similarly, the decrease of PI(3,4)P2 production reduced the FilGAP localization. Our results suggest that FilGAP localizes to invadopodia through its PH domain binding to PI(3,4)P2 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)P2.

Isotoosendanin exerts inhibition on triple-negative breast cancer through abrogating TGF-ß-induced epithelial-mesenchymal transition via directly targeting TGFßR1.

As the most aggressive breast cancer, triple-negative breast cancer (TNBC) is still incurable and very prone to metastasis. The transform growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of TNBC. This study reported that a natural compound isotoosendanin (ITSN) reduced TNBC metastasis by inhibiting TGF-ß-induced EMT and the formation of invadopodia. ITSN can directly interact with TGF-ß receptor type-1 (TGFßR1) and abrogated the kinase activity of TGFßR1, thereby blocking the TGF-ß-initiated downstream signaling pathway. Moreover, the ITSN-provided inhibition on metastasis obviously disappeared in TGFßR1-overexpressed TNBC cells in vitro as well as in mice bearing TNBC cells overexpressed TGFßR1. Furthermore, Lys232 and Asp351 residues in the kinase domain of TGFßR1 were found to be crucial for the interaction of ITSN with TGFßR1. Additionally, ITSN also improved the inhibitory efficacy of programmed cell death 1 ligand 1 (PD-L1) antibody for TNBC in vivo via inhibiting the TGF-ß-mediated EMT in the tumor microenvironment. Our findings not only highlight the key role of TGFßR1 in TNBC metastasis, but also provide a leading compound targeting TGFßR1 for the treatment of TNBC metastasis. Moreover, this study also points out a potential strategy for TNBC treatment by using the combined application of anti-PD-L1 with a TGFßR1 inhibitor.