The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function.

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

Tks adaptor proteins at a glance.

Tyrosine kinase substrate (Tks) adaptor proteins are considered important regulators of various physiological and/or pathological processes, particularly cell migration and invasion, and cancer progression. These proteins contain PX and SH3 domains, and act as scaffolds, bringing membrane and cellular components in close proximity in structures known as invadopodia or podosomes. Tks proteins, analogous to the related proteins p47phox, p40phox and NoxO1, also facilitate local generation of reactive oxygen species (ROS), which aid in signaling at invadopodia and/or podosomes to promote their activity. As their name suggests, Tks adaptor proteins are substrates for tyrosine kinases, especially Src. In this Cell Science at a Glance article and accompanying poster, we discuss the known structural and functional aspects of Tks adaptor proteins. As the science of Tks proteins is evolving, this article will point out where we stand and what still needs to be explored. We also underscore pathological conditions involving these proteins, providing a basis for future research to develop therapies for treatment of these diseases.

ADAM12 induction by Twist1 promotes tumor invasion and metastasis via regulation of invadopodia and focal adhesions.

The Twist1 transcription factor promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) and invadopodia-mediated extracellular matrix (ECM) degradation. The critical transcription targets of Twist1 for mediating these events remain to be uncovered. Here, we report that Twist1 strongly induces expression of a disintegrin and metalloproteinase 12 (ADAM12). We observed that the expression levels of Twist1 mRNA and ADAM12 mRNA are tightly correlated in human breast tumors. Knocking down ADAM12 blocked cell invasion in a 3D mammary organoid culture. Suppression of ADAM12 also inhibited Twist1-induced tumor invasion and metastasis in human breast tumor xenografts, without affecting primary tumor formation. Mechanistically, knockdown of ADAM12 in breast cancer cells significantly reduced invadopodia formation and matrix degradation, and simultaneously increased overall cell adhesion to the ECM. Live-imaging analysis showed that knockdown of ADAM12 significantly inhibited focal adhesion turnover. Mechanistically, both the disintegrin and metalloproteinase domains of ADAM12 are required for its function at invadopodia, whereas the metalloproteinase domain is dispensable for its function at focal adhesions. Taken together, these data suggest that ADAM12 plays a crucial role in tumor invasion and metastasis by regulating both invadopodia and focal adhesions.

Assembly and biological role of podosomes and invadopodia.

Regulated tissue invasion via motile and lytic events is critical for physiological processes such as immune system function and inflammatory responses, wound healing, and organ development, but pathological subversion of this process drives tumour cell invasion and metastasis. Cell migration and invasion require the integration of several processes that include: first, the local modulation of cytoskeleton structure and contractile forces; second, the turnover of substrate adhesions and their associated microfilaments; and third, the generation of specialised, transient domains that mediate the protease-dependent focal degradation of the extracellular matrix. Recent work has re-discovered prominent actin-based cellular structures, termed invadopodia and podosomes, as unique structural and functional modules through which major invasive mechanisms are regulated. The stage is now set to unravel their roles in the physiology and pathology of tissue plasticity and repair.

Src-dependent Tks5 phosphorylation regulates invadopodia-associated invasion in prostate cancer cells.

BACKGROUND: The Src tyrosine kinase substrate and adaptor protein Tks5 had previously been implicated in the invasive phenotype of normal and transformed cell types via regulation of cytoskeletal structures called podosomes/invadopodia. The role of Src-Tks5 signaling in invasive prostate cancer, however, had not been previously evaluated. METHODS: We measured the relative expression of Tks5 in normal (n = 20) and cancerous (n = 184, from 92 patients) prostate tissue specimens by immunohistochemistry using a commercially available tumor microarray. We also manipulated the expression and activity of wild-type and mutant Src and Tks5 constructs in the LNCaP and PC-3 prostate cancer cell lines in order to ascertain the role of Src-Tks5 signaling in invadopodia development, matrix-remodeling activity, motility, and invasion. RESULTS: Our studies demonstrated that Src was activated and Tks5 upregulated in high Gleason score prostate tumor specimens and in invasive prostate cancer cell lines. Remarkably, overexpression of Tks5 in LNCaP cells was sufficient to induce invadopodia formation and associated matrix degradation. This Tks5-dependent increase in invasive behavior further depended on Src tyrosine kinase activity and the phosphorylation of Tks5 at tyrosine residues 557 and 619. In PC-3 cells we demonstrated that Tks5 phosphorylation at these sites was necessary and sufficient for invadopodia-associated matrix degradation and invasion. CONCLUSIONS: Our results suggest a general role for Src-Tks5 signaling in prostate tumor progression and the utility of Tks5 as a marker protein for the staging of this disease.

Disruption of the podosome adaptor protein TKS4 (SH3PXD2B) causes the skeletal dysplasia, eye, and cardiac abnormalities of Frank-Ter Haar Syndrome.

Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.

The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.

Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Cell migration and invasion in human disease: the Tks adaptor proteins.

Cell invasion plays a central role in a wide variety of biological phenomena and is the cause of tumour growth and metastasis. Understanding the biochemical mechanisms that control cell invasion is one of the major goals of our laboratory. Podosomes and invadopodia are specialized cellular structures present in cells with physiological or pathological invasive behaviours. These transient structures are localized at the ventral cell surface, contain an array of different proteins and facilitate cell-substrate adhesion, as well as the local proteolytic activity necessary for extracellular matrix remodelling and subsequent cellular invasion. We have shown previously that the adaptor proteins and Src substrates Tks4 and Tks5 are required for podosome and invadopodia formation, for cancer cell invasion in vitro, and for tumour growth in vivo. We have also defined a role for the Tks-mediated generation of ROS (reactive oxygen species) in both podosome and invadopodia formation, and invasive behaviour. Tks4 and Tks5 are also required for proper embryonic development, probably because of their roles in cell migration. Finally, we recently implicated podosome formation as part of the synthetic phenotype of vascular smooth muscle cells. Inhibitors of podosome and invadopodia formation might have utility in the treatment of vascular diseases and cancer. We have therefore developed a high-content cell-based high-throughput screening assay that allows us to identify inhibitors and activators of podosome/invadopodia formation. We have used this assay to screen for small-molecule inhibitors and defined novel regulators of invadopodia formation. In the present paper, I review these recent findings.

Megakaryocytes form linear podosomes devoid of digestive properties to remodel medullar matrix.

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.

Podosomal proteins as causes of human syndromes: a role in craniofacial development?

Podosomes and invadopodia are actin-rich protrusions of the plasma membrane important for matrix degradation and cell migration. Most of the information in this field has been obtained in cancer cells, where the presence of invadopodia has been related to increased invasiveness and metastatic potential. The importance of the related podosome structure in other pathological or physiological processes that require cell invasion is relatively unexplored. Recent evidence indicates that essential components of podosomes are responsible for several human syndromes, some of which are characterized by serious developmental defects involving the craniofacial area, skeleton and heart, and very poor prognosis. Here we will review them and discuss the possible role of podosomes as a player in correct embryo development.

Serine-Threonine Kinase TAO3-Mediated Trafficking of Endosomes Containing the Invadopodia Scaffold TKS5α Promotes Cancer Invasion and Tumor Growth.

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.

Tks5 recruits AFAP-110, p190RhoGAP, and cortactin for podosome formation.

Podosome formation in vascular smooth muscle cells is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the molecular mechanism that underlies their specific recruitment to podosomes remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. We show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5 mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. By expressing a catalytically inactive point mutant and by siRNA-mediated expression knock-down we also provide evidence that p190RhoGAP is required for podosome formation. Together our findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation.

Crosstalk between invadopodia and the extracellular matrix.

The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native collagen-I, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

The Translational Research Working Group developmental pathway for anticancer agents (drugs or biologics).

The Translational Research Working Group (TRWG) was created as a national initiative to evaluate the current status of the National Cancer Institute's investment in translational research and envision its future. The TRWG conceptualized translational research as a set of six developmental processes or pathways focused on various clinical goals. One of those pathways describes the development of agents-both small molecules and biologics-for the treatment and prevention of cancer. The Agents Developmental Pathway was conceived not as a comprehensive description of the corresponding real-world processes, but rather as a tool designed to facilitate movement of an agent through the translational process to the point where it can begin definitive clinical testing. This article presents the Agents Developmental Pathway and discusses key challenges associated with the processes described.

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo.

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.

Invadosomes are coming: new insights into function and disease relevance.

Invadopodia and podosomes are discrete, actin-based molecular protrusions that form in cancer cells and normal cells, respectively, in response to diverse signaling pathways and extracellular matrix cues. Although they participate in a host of different cellular processes, they share a common functional theme of controlling pericellular proteolytic activity, which sets them apart from other structures that function in migration and adhesion, including focal adhesions, lamellipodia, and filopodia. In this review, we highlight research that explores the function of these complex structures, including roles for podosomes in embryonic and postnatal development, in angiogenesis and remodeling of the vasculature, in maturation of the postsynaptic membrane, in antigen sampling and recognition, and in cell-cell fusion mechanisms, as well as the involvement of invadopodia at multiple steps of the metastatic cascade, and how all of this may apply in the treatment of human disease states. Finally, we explore recent research that implicates a novel role for exosomes and microvesicles in invadopodia-dependent and invadopodia-independent mechanisms of invasion, respectively.

SRC Increases MYC mRNA Expression in Estrogen Receptor-Positive Breast Cancer via mRNA Stabilization and Inhibition of p53 Function.

The transcription factor gene MYC is important in breast cancer, and its mRNA is maintained at a high level even in the absence of gene amplification. The mechanism(s) underlying increased MYC mRNA expression is unknown. Here, we demonstrate that MYC mRNA was stabilized upon estrogen stimulation of estrogen receptor-positive breast cancer cells via SRC-dependent effects on a recently described RNA-binding protein, IMP1 with an N-terminal deletion (ΔN-IMP1). We also show that loss of the tumor suppressor p53 increased MYC mRNA levels even in the absence of estrogen stimulation. However, in cells with wild-type p53, SRC acted to overcome p53-mediated inhibition of estrogen-stimulated cell cycle entry and progression. SRC thus promotes cell proliferation in two ways: by stabilizing MYC mRNA and by inhibiting p53 function. Since estrogen receptor-positive breast cancers typically express wild-type p53, these studies establish a rationale for p53 status to be predictive for effective SRC inhibitor treatment in this subtype of breast cancer.

Induction of anaplastic lymphoma kinase (ALK) as a novel mechanism of EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models.

Head and neck squamous cell carcinoma (HNSCC) currently only has one FDA-approved cancer intrinsic targeted therapy, the epidermal growth factor receptor (EGFR) inhibitor cetuximab, to which only approximately 10% of tumors are sensitive. In order to extend therapy options, we subjected patient-derived HNSCC cells to small-molecule inhibitor and siRNA screens, first, to find effective combination therapies with an EGFR inhibitor, and second, to determine a potential mechanistic basis for repurposing the FDA approved agents for HNSCC. The combinations of EGFR inhibitor with anaplastic lymphoma kinase (ALK) inhibitors demonstrated synergy at the highest ratio in our cohort, 4/8 HNSCC patients' derived tumor cells, and this corresponded with an effectiveness of siRNA targeting ALK combined with the EGFR inhibitor gefitinib. Co-targeting EGFR and ALK decreased HNSCC cell number and colony formation ability and increased annexin V staining. Because ALK expression is low and ALK fusions are infrequent in HNSCC, we hypothesized that gefitinib treatment could induce ALK expression. We show that ALK expression was induced in HNSCC patient-derived cells both in 2D and 3D patient-derived cell culture models, and in patient-derived xenografts in mice. Four different ALK inhibitors, including two (ceritinib and brigatinib) FDA approved for lung cancer, were effective in combination with gefitinib. Together, we identified induction of ALK by EGFR inhibitor as a novel mechanism potentially relevant to resistance to EGFR inhibitor, a high ratio of response of HNSCC patient-derived tumor cells to a combination of ALK and EGFR inhibitors, and applicability of repurposing ALK inhibitors to HNSCC that lack ALK aberrations.

Cell fusion potentiates tumor heterogeneity and reveals circulating hybrid cells that correlate with stage and survival.

High lethality rates associated with metastatic cancer highlight an urgent medical need for improved understanding of biologic mechanisms driving metastatic spread and identification of biomarkers predicting late-stage progression. Numerous neoplastic cell intrinsic and extrinsic mechanisms fuel tumor progression; however, mechanisms driving heterogeneity of neoplastic cells in solid tumors remain obscure. Increased mutational rates of neoplastic cells in stressed environments are implicated but cannot explain all aspects of tumor heterogeneity. We present evidence that fusion of neoplastic cells with leukocytes (for example, macrophages) contributes to tumor heterogeneity, resulting in cells exhibiting increased metastatic behavior. Fusion hybrids (cells harboring hematopoietic and epithelial properties) are readily detectible in cell culture and tumor-bearing mice. Further, hybrids enumerated in peripheral blood of human cancer patients correlate with disease stage and predict overall survival. This unique population of neoplastic cells provides a novel biomarker for tumor staging, as well as a potential therapeutic target for intervention.

The role of Tks adaptor proteins in invadopodia formation, growth and metastasis of melanoma.

Metastatic cancer cells are characterized by their ability to degrade and invade through extracellular matrix. We previously showed that the Tks adaptor proteins, Tks4 and Tks5, are required for invadopodia formation and/or function in Src-transformed fibroblasts and a number of human cancer cell types. In this study, we investigated the role of Tks adaptor proteins in melanoma cell invasion and metastasis. Knockdown of either Tks4 or Tks5 in both mouse and human melanoma cell lines resulted in a decreased ability to form invadopodia and degrade extracellular matrix. In addition, Tks-knockdown melanoma cells had decreased proliferation in a 3-dimensional type l collagen matrix, but not in 2-dimensional culture conditions. We also investigated the role of Tks proteins in melanoma progression in vivo using xenografts and experimental metastasis assays. Consistent with our in vitro results, reduction of Tks proteins markedly reduced subcutaneous melanoma growth as well as metastatic growth in the lung. We explored the clinical relevance of Tks protein expression in human melanoma specimens using a tissue microarray. Compared to non-malignant nevi, both Tks proteins were highly expressed in melanoma tissues. Moreover, metastatic melanoma cases showed higher expression of Tks5 than primary melanoma cases. Taken together, these findings suggest the importance of Tks adaptor proteins in melanoma growth and metastasis in vivo, likely via functional invadopodia formation.