The Converging Roles of Cholesterol and C-Terminal Src Kinase in the Regulation of Extracellular Matrix Degradation at Invadopodia

Metastasis, the leading cause of mortality in cancer patients, is the dissemination of cancer cells from the primary tumour and spread to the distant sites of the body. This is a complex process during which tumour cells need to overcome several natural barriers to gain entry into the bloodstream and thus allow formation of secondary tumour in distant location. In the last two decades, much effort has been focused on showing that tumour cells use specialized actin-based membrane protrusions termed invadopodia to perform matrix degradation. Invadopodia gain their protrusive capacity combining the mechanical force of actin polymerization with the chemical activity of matrix degradation. As such, invadopodia are F-actin-rich structures enriched in integrins, tyrosine kinases signalling machinery, soluble and membrane proteases, including matrix metalloproteases (MMPs), and actin-associated proteins. How all these components are specifically recruited to the ECM degradation sites has not been fully clarified yet. An emerging model describes invadopodia as dynamic cellular platforms where the signalling, membrane trafficking and cytoskeleton remodelling converge upstream of ECM degradation at spatially confined cholesterol-rich membrane compartments. Despite the field of invadopodia biogenesis and function is still a very recent, it is witnessing an increasing interest and an increasing number of molecular players have been identified in the last two decades. Although the existence of the invadopodia-like structures in vivo settings still needs to be determined, invadopodia represent powerful experimental paradigm to study the tight integration between the signalling, the membrane trafficking and cytoskeleton remodelling upstream of ECM degradation, the rate-limiting step in cell invasion, and might provide better understanding of cancer cell invasion and metastasis. In order to investigate the cholesterol-rich lipid raft feature of invadopodia, I followed three lines. First, I showed that inhibition of cholesterol formation at penultimate step of its biosynthesis, and subsequent accumulation of desmosterol, blocks formation and function of invadopodia, thus demonstrating that the central role of cholesterol is connected to its presence in functional lipid rafts. In the second approach, I found that the SFKs inhibitory kinase Csk is a negative regulator of invadopodia-mediated ECM degradation and its role depends on the localization in the cholesterol-rich lipid rafts. Finally, I demonstrated that free cholesterol-dependent ARF6-associated recycling pathway might be involved in the trafficking to invadopodia, while the ARF6-pathway constituents are localized at ECM degradation sites and ARF6-specific cargo CD 147 is recycled to invadopodia. Taken together, my findings provide novel insight towards the elucidation of invadopodia as specialized cholesterol-dependent membrane domains where signal transduction and membrane trafficking events might be temporally and spatially confined

MT1-MMP Mediates the Migratory and Tumourigenic Potential of Breast Cancer Cells via Non-Proteolytic Mechanisms

Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that affects cell function via proteolytic and non-proteolytic mechanisms such as promoting degradation of the extracellular matrix (ECM) or augmentation of cell migration and viability, respectively. MT1-MMP has been implicated in metastatic progression ostensibly due to its ability to degrade ECM components and to allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) that inhibit substrate catalysis as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete. To further our understanding of MT1-MMP function, MCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, I analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. Matrigel 3D cell culture and avian embryos were also utilized to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenicity and extravasation efficiency in vivo. In 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenicity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein. This study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo

The Cytoplasmic Domain of Membrane-type 1 Matrix Metalloproteinase is Required for its Survival-Promoting, but not its Migration-Promoting Function in MCF-7 Breast Cancer Cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a multifunctional protease that degrades proteins during cell migration, and influences cell survival. Both the protein localization and signal transduction capabilities of MT1-MMP depend on its cytoplasmic domain (CD), indicative of a diverse regulatory function. The effects of CD mutations on cell migration and survival were examined by ectopically expressing MT1-MMP variants in MCF-7 cells. CD alteration by substitution or deletion did not abolish the migration-promoting effects of MT1-MMP, but did decrease cell survival and increase apoptosis. Expression of CD-altered MT1-MMP resulted in a protrusive cell morphology in 3D culture that was lost upon serum starvation. MT1-MMP expression in a chicken embryo tumour model resulted in vascularization of MCF-7 tumours; a phenotype that was partially maintained following expression of MT1-MMP CD variants. These results suggest that the CD regulates MT1-MMP localization in a manner required for cell survival, but is dispensable for cell migration

Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK

Summary Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) 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, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR 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

5′-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5′-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2–Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease

Tumor cellular and microenvironmental cues controlling invadopodia formation

During the metastatic progression, invading cells might achieve degradation and subsequent invasion into the extracellular matrix (ECM) and the underlying vasculature using invadopodia, F-actin-based and force-supporting protrusive membrane structures, operating focalized proteolysis. Their formation is a dynamic process requiring the combined and synergistic activity of ECM-modifying proteins with cellular receptors, and the interplay with factors from the tumor microenvironment (TME). Significant advances have been made in understanding how invadopodia are assembled and how they progress in degradative protrusions, as well as their disassembly, and the cooperation between cellular signals and ECM conditions governing invadopodia formation and activity, holding promise to translation into the identification of molecular targets for therapeutic interventions. These findings have revealed the existence of biochemical and mechanical interactions not only between the actin cores of invadopodia and specific intracellular structures, including the cell nucleus, the microtubular network, and vesicular trafficking players, but also with elements of the TME, such as stromal cells, ECM components, mechanical forces, and metabolic conditions. These interactions reflect the complexity and intricate regulation of invadopodia and suggest that many aspects of their formation and function remain to be determined. In this review, we will provide a brief description of invadopodia and tackle the most recent findings on their regulation by cellular signaling as well as by inputs from the TME. The identification and interplay between these inputs will offer a deeper mechanistic understanding of cell invasion during the metastatic process and will help the development of more effective therapeutic strategies

Regulation of MT1-MMP Activity through Its Association with ERMs

Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM molecules. At the plasma membrane MT1-MMP autoprocessing is severely dependent on ERM association and seems to be the dominant regulator of the enzyme collagenolytic activity. This newly characterized MT1-MMP/ERM association can thus be of relevance for tumor cell invasion.This work has been supported by grants BFU2014-55478-R, REDIEX. SAF2015-71231-REDT and BIO2017-86500-R from Ministerio Español de Economía y Competitividad (MINECO) and by a grant from Fundación Ramón Areces “Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014” to M.Y.-M. H.S. was supported by a FPI-UAM fellowship. The CNIC is supported by the Ministry of Ciencia, Innovacion y Universidades and the Pro CNIC Foundation, is a Severo Ochoa Center of Excellence (SEV-2015-0505), also supported by European Regional Development Fund (FEDER) “Una manera de hacer Europa”.S

Nucleobindin-1 modulates extracellular matrix remodeling by promoting intra-Golgi trafficking of matrix metalloproteinase 2

Matrix metalloproteases (MMPs) play a crucial role in tissue homeostasis. Profuse literature has studied their roles in cell migration and tissue invasion during cancer metastasis, as well as in inflammatory processes. Although the literature covering MMPs function is abundant, the intracellular trafficking of these proteins remains poorly understood. The aim of the present work was to identify the molecular mechanism of intracellular trafficking of MMPs, with particular focus on MMP2. A novel mass spectrometry approach revealed nucleobindin-1 (NUCB1), a major regulator of Ca2+ homeostasis at the Golgi, as a potential candidate for the regulation of MMP transport. Using a synchronized cargo trafficking assay, it was possible to demonstrate that in the absence of NUCB1 the intracellular trafficking of MMP2 is delayed. Moreover, this work reveals that NUCB1-dependent MMP2 trafficking is restricted to the Golgi, exclusively delaying its intra-Golgi trafficking at the cis compartment and, as a consequence, decreasing MMP2 mediated cell migration and matrix invasion. Furthermore, my findings show that not only MMP2, but also MT1-MMP intra-Golgi trafficking is impaired, implying that this mechanism could also influence the trafficking of other MMPs. Interestingly, experiments performed with a NUCB1 Ca2+-binding deficient mutant showed that Ca2+ is required, both for the interaction, as well as for proper MMP2 trafficking, suggesting that a specific impairment of cis-Golgi Ca2+ homeostasis, rather than an overall Ca2+ deficiency, is essential for proper MMP2 intra-Golgi trafficking. Taken together the results of this thesis contributed to enlighten the mechanism of MMP2 intracellular trafficking by identifying NUCB1 as a critical player in MMP transport. Importantly, this work highlights the requirement of Ca2+ for proper trafficking, not just at the TGN, as has been documented, but also at the cis-Golgi. Although this is a big step towards the understanding of MMP intracellular trafficking, further investigations are required to gain a better understanding of the retention mechanism of NUCB1 at the cis-Golgi lumen and a deeper insight into the regulation of intra-Golgi protein trafficking

Localisation of MT1-MMP to motility-associated structures

MT1-MMP is a crucial enzyme for cellular invasion in tissues, and its polarised cell surface localisation is thought to be an important regulatory mechanism. In this thesis, the mechanisms regulating localisation of MT1-MMP to motility-associated structures were investigated. Using a series of MT1-MMP domain deletion mutants, it was found that an eight amino acid region in the catalytic domain called the “MT-Loop” is involved in localisation of the enzyme to matrix attachment sites. A previously described mechanism by which MT1-MMP localises to lamellipodia is association with CD44. Interestingly, knockdown of the CD44 in HT-1080 cells increased MT1-MMP localisation to the matrix attachment sites, whereas CD44 overexpression in COS-7 cells decreased this localisation. These results indicate that CD44-mediated localisation to lamellipodia may compete with the localisation to the matrix attachment sites. One of the previously reported mechanisms localising MT1-MMP to the matrix attachment sites is by targeting the enzyme to cortactin-mediated invadopodia structures. However, silencing of the cortactin gene in HT-1080 cells unexpectedly enhanced this localisation, suggesting that invadopodia may not be required. For MT1-MMP to appear on the cell surface, the newly expressed molecules need to be trafficked from the trans-Golgi network to the plasma membrane by kinesin motor proteins (KIFs). 17 KIFs were tested for their involvement in the intracellular trafficking of MT1-MMP-containing vesicles. Silencing of the KIF3A or KIF13A genes markedly decreased MT1-MMP-dependent gelatin and collagen film degradation, whereas silencing of the KIF9 and KIF1C genes enhanced degradation of the underlying matrix. These and other data presented in this thesis suggest that KIF3A, KIF13A, KIF9 and KIF1C may be involved in vesicle trafficking of MT1-MMP to different areas of the plasma membrane and regulate spatial localisation of the enzyme. Taken together, these findings indicate that polarised cell surface localisation of MT1-MMP is regulated by multiple mechanisms, which may be important for the enzyme to promote cellular invasion in different microenvironments

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases