Regulates Invadopodia Formation Research Articles

Ezrin promotes invasion and metastasis of hepatocellular carcinoma by regulating invadopodia formation

Ezrin promotes invasion and metastasis of hepatocellular carcinoma by regulating invadopodia formation

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42

Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.

Invadopodia, a Kingdom of Non-Receptor Tyrosine Kinases.

Non-receptor tyrosine kinases (NRTKs) are crucial mediators of intracellular signaling and control a wide variety of processes such as cell division, morphogenesis, and motility. Aberrant NRTK-mediated tyrosine phosphorylation has been linked to various human disorders and diseases, among them cancer metastasis, to which no treatment presently exists. Invasive cancer cells leaving the primary tumor use invadopodia, feet-like structures which facilitate extracellular matrix (ECM) degradation and intravasation, to escape the primary tumor and disseminate into distant tissues and organs during metastasis. A major challenge in metastasis research is to elucidate the molecular mechanisms and signaling pathways underlying invadopodia regulation, as the general belief is that targeting these structures can potentially lead to the eradication of cancer metastasis. Non-receptor tyrosine kinases (NRTKs) play a central role in regulating invadopodia formation and function, but how they coordinate the signaling leading to these processes was not clear until recently. Here, we describe the major NRTKs that rule invadopodia and how they work in concert while keeping an accurate hierarchy to control tumor cell invasiveness and dissemination.

Role of Clathrin Light Chains in Regulating Invadopodia Formation.

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells

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.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Cross-Talk between Receptor Tyrosine Kinases AXL and ERBB3 Regulates Invadopodia Formation in Melanoma Cells.

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.

Targeting invadopodia for blocking breast cancer metastasis.

Dissemination of cancer cells from the primary tumor and their spread to distant sites of the body is the leading cause of mortality in metastatic cancer patients. Metastatic cancer cells invade surrounding tissues and blood vessels by forming F-actin-rich protrusions known as invadopodia, which degrade the extracellular matrix and enable invasion of tumor cells through it. Invadopodia have now been observed in vivo, and recent evidence demonstrates direct molecular links between assembly of invadopodia and cancer metastasis in both mouse models and in human patients. While significant progress has been achieved in the last decade in understanding the molecular mechanisms and signaling pathways regulating invadopodia formation and function, the application of this knowledge to development of prognostic and therapeutic approaches for cancer metastasis has not been discussed before. Here, we provide a detailed overview of current prognostic markers and tests for cancer metastasis and discuss their advantages, disadvantages, and their predicted efficiency. Using bioinformatic patient database analysis, we demonstrate, for the first time, a significant correlation between invadopodia-associated genes to breast cancer metastasis, suggesting that invadopodia could be used as both a prognostic marker and as a therapeutic target for blocking cancer metastasis. We include here a novel network interaction map of invadopodia-associated proteins with currently available inhibitors, demonstrating a central role for the recently identified EGFR-Pyk2-Src-Arg-cortactin invadopodial pathway, to which re-purposing of existent inhibitors could be used to block breast cancer metastasis. We then present an updated overview of current cancer-related clinical trials, demonstrating the negligible number of trials focusing on cancer metastasis. We also discuss the difficulties and complexity of performing cancer metastasis clinical trials, and the possible development of anti-metastasis drug resistance when using a prolonged preventive treatment with invadopodia inhibitors. This review presents a new perspective on invadopodia-mediated tumor invasiveness and may lead to the development of novel prognostic and therapeutic approaches for cancer metastasis.

Phosphatidylinositol 4-kinase IIβ negatively regulates invadopodia formation and suppresses an invasive cellular phenotype.

The type II phosphatidylinositol 4-kinase (PI4KII) enzymes synthesize the lipid phosphatidylinositol 4-phosphate (PI(4)P), which has been detected at the Golgi complex and endosomal compartments and recruits clathrin adaptors. Despite common mechanistic similarities between the isoforms, the extent of their redundancy is unclear. We found that depletion of PI4KIIα and PI4KIIβ using small interfering RNA led to actin remodeling. Depletion of PI4KIIβ also induced the formation of invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). Depletion of PI4KII isoforms also differentially affected trans-Golgi network (TGN) pools of PI(4)P and post-TGN traffic. PI4KIIβ depletion caused increased MT1-MMP trafficking to invasive structures at the plasma membrane and was accompanied by reduced colocalization of MT1-MMP with membranes containing the endosomal markers Rab5 and Rab7 but increased localization with the exocytic Rab8. Depletion of PI4KIIβ was sufficient to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines. Mining oncogenomic databases revealed that loss of the PI4K2B allele and underexpression of PI4KIIβ mRNA are associated with human cancers. This finding supports the cell data and suggests that PI4KIIβ may be a clinically significant suppressor of invasion. We propose that PI4KIIβ synthesizes a pool of PI(4)P that maintains MT1-MMP traffic in the degradative pathway and suppresses the formation of invadopodia.

Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM) biology and represent novel sources of biomarkers that are detectable in the peripheral circulation. Despite this notionally non-invasive approach to assess GBM tumours in situ, a comprehensive GBM EV protein signature has not been described. Here, EVs secreted by six GBM cell lines were isolated and analysed by quantitative high-resolution mass spectrometry. Overall, 844 proteins were identified in the GBM EV proteome, of which 145 proteins were common to EVs secreted by all cell lines examined; included in the curated EV compendium (Vesiclepedia_559; http://microvesicles.org). Levels of 14 EV proteins significantly correlated with cell invasion (invadopodia production; r2 > 0.5, p < 0.05), including several proteins that interact with molecules responsible for regulating invadopodia formation. Invadopodia, actin-rich membrane protrusions with proteolytic activity, are associated with more aggressive disease and are sites of EV release. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-β1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours compared to normal brain in silico, with common functions relating to actin polymerisation and endosomal sorting. We also show that Cavitron Ultrasonic Surgical Aspirator (CUSA) washings are a novel source of brain tumour-derived EVs, demonstrated by particle tracking analysis, TEM and proteome profiling. Quantitative proteomics corroborated the high levels of proposed invasion-related proteins in EVs enriched from a GBM compared to low-grade astrocytoma tumour. Large-scale clinical follow-up of putative biomarkers, particularly the proposed survival marker annexin A1, is warranted.

Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells

The ability of cancer cells to degrade the extracellular matrix and invade interstitial tissues contributes to their metastatic potential. We recently showed that overexpression of sorting nexin 9 (SNX9) leads to increased cell invasion and metastasis in animal models, which correlates with increased SNX9 protein expression in metastases from human mammary cancers. Here, we report that SNX9 expression is reduced relative to neighboring normal tissues in primary breast tumors, and progressively reduced in more aggressive stages of non-small-cell lung cancers. We show that SNX9 is localized at invadopodia where it directly binds the invadopodia marker TKS5 and negatively regulates invadopodia formation and function. SNX9 depletion increases invadopodia number and the local recruitment of MT1-MMP by decreasing its internalization. Together, these effects result in increased localized matrix degradation. We further identify SNX9 as a Src kinase substrate and show that this phosphorylation is important for SNX9 activity in regulating cell invasion, but is dispensable for its function in regulating invadopodia. The diversified changes associated with SNX9 expression in cancer highlight its importance as a central regulator of cancer cell behavior.

Abstract 3155: Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma

Abstract Invadopodia are actin-based proteolytic structures which facilitate cancer cell invasion and metastasis. While it was previously believed that invadopodia are not required for tumor cell growth, based on studies in monolayer cell culture, more recently we have noted a role for invadopodia proteins in growth in more physiological 3-dimensional (3D) extracellular matrix contexts. From previous work in our lab, the Tks adaptor proteins with 5 (Tks5) and 4 (Tks4) SH3 domains, respectively, were identified as Src kinase substrates and both are essential molecules for functional invadopodia formation in mouse and human cancer cells. Here, we assess the roles of the Tks adaptors in melanoma tumor growth and invasion both in vivo and in vitro. To address the mechanism as to how the Tks adaptors control tumor growth and invasion, in vitro 3D growth assays in collagen I were performed. Knockdown of either Tks4 or Tks5 in C8161.9 or A375 melanoma cells decreased 3D growth in collagen I, a process that also appeared to be dependent upon metalloproteinase (MMP) activity. Interestingly, knockdown of either Tks4 or Tks5 in A375 melanoma cells decreased cell-surface expression of MT1-MMP, a key protease involved in tumor growth and metastasis. In vivo tumor growth and metastasis assays in mice using human and mouse melanoma cells depleted of Tks4 or Tks5 by shRNA revealed roles for the Tks adaptors in both the size and the number of lung metastases. Finally, analysis of a human melanoma tissue array with a Tks5 antibody, and RNAseq data, indicates that Tks5 expression is increased in lymph node and other metastases in comparison to nevi and primary sites. These data suggest that the Tks adaptors control melanoma tumor growth and invasion by regulating invadopodia formation and perhaps also cell surface expression of MT1-MMP, and thus contribute to melanoma progression. Citation Format: Shinji Iizuka, Christine M. Gould, Matthew D. Buschman, Diaz Begoña, Christopher Abdullah, Sara Courtneidge. Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3155. doi:10.1158/1538-7445.AM2014-3155

SNARE-dependent interaction of Src, EGFR and β1 integrin regulates invadopodia formation and tumor cell invasion

Acquisition of an invasive phenotype is prerequisite for tumor metastasis. Degradation of the extracellular matrix (ECM), and subsequent invasion by tumor cells, is mediated, in part, through subcellular structures called invadopodia. Src-dependent cytoskeletal rearrangements are required to form invadopodia, and here we identify an association between Src, epidermal growth factor receptor (EGFR), and β1 integrin that facilitates invadopodia formation. The association of Src, EGFR and β1 integrin is dependent upon membrane traffic that is mediated by syntaxin13 (officially known as STX12) and SNAP23; a similar dependence on these two SNARE proteins was observed for invadopodium-based matrix degradation and cell invasion. Inhibition of SNARE function impaired the delivery of Src and EGFR to developing invadopodia, as well as the β1-integrin-dependent activation of Src and phosphorylation of EGFR on Tyr residue 845. We also identified an association between SNAP23 and β1 integrin, and inhibition of β1 integrin increased this association, whereas the interaction between syntaxin13 and SNAP23 was reduced. The results suggest that SNARE-dependent trafficking is regulated, in part, by β1 integrin and is required for the delivery of Src and EGFR to sites of invadopodia formation in order to support tumor cell invasion.

Polarised apical-like intracellular sorting and trafficking regulates invadopodia formation and degradation of the extracellular matrix in cancer cells

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities.We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes.To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation.Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes.

Abstract 495: Overexpression of TIS21/BTG2/PC3 inhibits formation of invadopodia in the highly metastatic breast cancer cells MDA-MB-231

Abstract Invasion of cancer cells depends on the proteolytic degradation of extracellular matrix and it is initiated by the actin-driven membrane protrusions, called invadopodia. However, the mechanisms underlying invadopodia formation remain largely unknown. In this study, we report that TIS21/BTG2/PC3, tumor suppressor gene, suppress invadopodia formation in the highly invasive breast cancer cells, MDA-MB-231. To understand downstream mechanisms regulating invadopodia formation by TIS21, generation of ROS (reactive oxygen species) by NADPH oxidase (Nox) was examined and observed that adenoviral transduction of TIS21 markedly attenuated generation of ROS. The transduction of TIS21 significantly inhibited mRNA expression of Nox1 and Nox4, but not p22phox. In contrast, formation of invadopodia was much higher in the TIS21KO-mouse embryo fibroblasts (TIS21−/- MEF) along with elevated expression of Nox1 and Nox4, compared with those of the wild type MEF. The data strongly suggest that TIS21 contributes inhibition of invadopodia formation through the regulation of Nox expression. Noticeably, transduction of TIS21 regulated reorganization of actin-assoicated protein and cytockelectal proteins such as F-actin and vimentin. Similarly, inhibition of Nox-derived ROS by using DPI reversed reorganization of the proteins. The above data clearly indicate that generation of Nox-derived ROS play a critical role in the distribution of actin-assoicated protein and cytockelectal proteins. Taken together, present report demonstrates, for the first time, that the TIS21/BTG2-ROS-linked cascades is required for formation of invadopodia in the highly invasive breast cancer cells and may provide insight into mechanisms of metastatic signaling in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 495. doi:1538-7445.AM2012-495

Regulation of invadopodia formation and activity by CD147

A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia.

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.

Abstract 4748: Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Abstract Degradation of extracellular matrix (ECM) that is present in the basement membrane and tumor stroma is essential for local invasion and formation of metastatic sites by malignant cancer cells. Invadopodia are ECM-degrading protrusions formed by a variety of invasive cancer cell types and are thought to function in cancer invasion and metastasis. Many components of invadopodia, such as proteins involved in actin polymerization, cell signaling, and ECM degradation, have been reported to date. However, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that produce D-3 phosphoinositides. PI3Ks mediate the signal transduction of extracellular stimuli and regulate diverse cellular events. Uncontrolled activation of the PI3K signaling pathway leads to several pathological phenomena, including tumorigenesis and tumor malignancy. We investigated the role of PI3K signaling during invadopodia formation. We found that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix was blocked by treatment with PI3K inhibitors or sequestration of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. Mammals have 8 PI3K enzymes that are subdivided into three general classes (I-III) on the basis of their domain structures and substrate specificities. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α is selectively involved in invadopodia formation. p110α is encoded by the PIK3CA gene, which is one of the most frequently amplified and mutated genes identified in human cancers. Clinical studies revealed that activating mutations of PIK3CA are associated with the development of invasive and metastatic phenotypes and poor patient prognosis. The expression of p110α with the activating mutations promoted invadopodia-mediated invasive activity. Furthermore, knocking down PDK1 and Akt, the downstream effectors of PI3K signaling, suppressed invadopodia formation induced by the p110α mutants. These data suggest that the PI3K signaling pathway via p110α regulates invadopodia-mediated invasion of breast cancer cells. Targeting p110α may be a potential therapeutic approach for controlling invasion of malignant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2011-4748

Autotaxin Promotes Cancer Invasion via the Lysophosphatidic Acid Receptor 4: Participation of the Cyclic AMP/EPAC/Rac1 Signaling Pathway in Invadopodia Formation

The ability of cancer cells to invade and metastasize is the major cause of death in cancer patients. Autotaxin (ATX) is a secreted lysophospholipase whose level of expression within tumors correlates strongly with their aggressiveness and invasiveness. ATX is the major enzyme involved in the production of lysophosphatidic acid (LPA), a phospholipid that is known to act mostly through its three first characterized receptors (LPA(1), LPA(2), and LPA(3)). Tumor cell invasion across tissue boundaries and metastasis are dependent on the capacity of invasive cancer cells to breach the basement membrane. This process can be initiated by the formation of the actin-rich cell protrusions, invadopodia. In this study, we show that ATX is implicated in the formation of invadopodia in various cancer cells types and this effect is dependent on the production of LPA. We further provide evidence that LPA(4) signaling in fibrosarcoma cells regulates invadopodia formation downstream of ATX, a process mediated through the activation of EPAC by cyclic AMP and subsequent Rac1 activation. Results using LPA(4) shRNA support the requirement of the LPA(4) receptor for cell invasion and in vivo metastasis formation. This work presents evidence that blocking the LPA receptor, LPA(4), in fibrosarcoma cells could provide an additional tool to improve the efficacy of treatment of metastasis in patients. Because LPA receptors and ATX are currently being targeted in preclinical trials, the current findings should stimulate future studies to evaluate the expression pattern and clinical outcome of LPA(4), together with other LPA receptors, in various cancer patients.