ADAMTS Proteases: Their Multifaceted Role in the Regulation of Cancer Metastasis.

Cancer leads to nearly 10 million deaths worldwide per year. The tumour microenvironment (TME) is fundamental for tumour growth and progression. A key component of the TME, the extracellular matrix (ECM) has recently become a focus of interest in cancer research. Dysregulation of ECM synthesis and proteolysis leads to uncontrolled tumour growth and metastasis. Matrix remodelling enzymes, secreted by cancer cells and stromal cells, modify the overall structure and organisation of ECM proteins, therefore influencing biochemical interactions, tissue integrity and tissue turnover. While A Disintegrin and Metalloproteinases (ADAMs)' and matrix metalloproteinases' role in cancer has been deeply investigated, other proteolytic enzymes, like ADAMs with thrombospondin(-like) motifs (ADAMTSs) have been gaining interest due to their roles in modulating cancer cell-ECM interactions and oncogenic signalling pathways. In this review, we will discuss the dysregulation of ADAMTSs in cancer and their roles in regulating cancer development and progression, via ECM remodelling and cell signalling modulation.

Overcoming Nutrient Stress: Integrin αvß3-Driven Metabolic Adaptation Supports Tumor Initiation.

Nutrient stress accompanies several stages of tumor progression, including metastasis formation. Metabolic reprogramming is a hallmark of cancer, and it has been associated with stress tolerance and anchorage-independent cell survival. Adaptive responses are required to support cancer cell survival under these conditions. In this issue of Cancer Research, Nam and colleagues showed that the extracellular matrix (ECM) receptor integrin ß3 was upregulated in lung cancer cells in response to nutrient starvation, resulting in increased cell survival that was independent from ECM binding. Delving into the molecular mechanisms responsible for this, the authors found that integrin ß3 promoted glutamine metabolism and oxidative phosphorylation (OXPHOS) by activating a Src/AMPK/PGC1α signaling pathway. Importantly, in vivo experiments confirmed that OXPHOS inhibition suppressed tumor initiation in an orthotopic model of lung cancer, while ß3 knockout completely abrogated tumor initiation. These observations indicate that targeting signaling pathways downstream of αvß3 could represent a promising therapeutic avenue to prevent lung cancer progression and metastasis. See related article by Nam et al., p. 1630.

Macropinocytosis at the crossroad between nutrient scavenging and metabolism in cancer.

Macropinocytosis (MP), the actin-dependent bulk uptake of extracellular fluids, plays a central role in nutrient scavenging, allowing cancer cells to sustain their growth in the hypoxic and nutrient-deprived microenvironment often found in solid tumours. The lack of soluble nutrients and several oncogenic signalling pathways, with RAS being the most studied, push MP-dependent internalisation of extracellular proteins, which are then digested in the lysosomes, replenishing the intracellular nutrient pools. This review will highlight recent advances in understanding how MP is regulated in hypoxic cancers, how it impinges on chemoresistance, and how different MP cargos facilitate tumour growth. Finally, I will highlight the crosstalk between MP and extracellular matrix receptors.

The extracellular matrix supports breast cancer cell growth under amino acid starvation by promoting tyrosine catabolism.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

The Interplay between Cell-Extracellular Matrix Interaction and Mitochondria Dynamics in Cancer.

The tumor microenvironment, in particular the extracellular matrix (ECM), plays a pivotal role in controlling tumor initiation and progression. In particular, the interaction between cancer cells and the ECM promotes cancer cell growth and invasion, leading to the formation of distant metastasis. Alterations in cancer cell metabolism is a key hallmark of cancer, which is often associated with alterations in mitochondrial dynamics. Recent research highlighted that, changes in mitochondrial dynamics are associated with cancer migration and metastasis-these has been extensively reviewed elsewhere. However, less is known about the interplay between the extracellular matrix and mitochondria functions. In this review, we will highlight how ECM remodeling associated with tumorigenesis contribute to the regulation of mitochondrial function, ultimately promoting cancer cell metabolic plasticity, able to fuel cancer invasion and metastasis.

Cross-Talk Between the Tumor Microenvironment, Extracellular Matrix, and Cell Metabolism in Cancer.

The extracellular matrix (ECM) is a complex network of secreted proteins which provides support for tissues and organs. Additionally, the ECM controls a plethora of cell functions, including cell polarity, migration, proliferation, and oncogenic transformation. One of the hallmarks of cancer is altered cell metabolism, which is currently being exploited to develop anti-cancer therapies. Several pieces of evidence indicate that the tumor microenvironment and the ECM impinge on tumor cell metabolism. Therefore, it is essential to understand the contribution of the complex 3D microenvironment in controlling metabolic plasticity and responsiveness to therapies targeting cell metabolism. In this mini-review, we will describe how the tumor microenvironment and cancer-associated fibroblasts dictate cancer cell metabolism, resulting in increased tumor progression. Moreover, we will define the cross-talk between nutrient signaling and the trafficking of the ECM receptors of the integrin family. Finally, we will present recent data highlighting the contribution of nutrient scavenging from the microenvironment to support cancer cells growth under nutrient starvation conditions.

Membrane dynamics in cell migration.

Migration of cells is required in multiple tissue-level processes, such as in inflammation or cancer metastasis. Endocytosis is an extremely regulated cellular process by which cells uptake extracellular molecules or internalise cell surface receptors. While the role of endocytosis of focal adhesions (FA) and plasma membrane (PM) turnover at the leading edge of migratory cells is wide known, the contribution of endocytic proteins per se in migration has been frequently disregarded. In this review, we describe the novel functions of the most well-known endocytic proteins in cancer cell migration, focusing on clathrin, caveolin, flotillins and GRAF1. In addition, we highlight the relevance of the macropinocytic pathway in amoeboid-like cell migration.

Extracellular matrix internalization links nutrient signalling to invasive migration.

Integrins are the key mediators of cell-extracellular matrix (ECM) interaction, linking the ECM to the actin cytoskeleton. Besides localizing at the cell surface, they can be internalized and transported back to the plasma membrane (recycled) or delivered to the late endosomes/lysosomes for degradation. We and others have shown that integrin can be endocytosed together with their ECM ligands. In this short review, I will highlight how extracellular protein (including ECM) endocytosis impinges on the activation of the mechanistic target of rapamycin (mTOR) pathway, a master regulator of cell metabolism and growth. This supports the intriguing hypothesis that ECM components may be considered as nutrient sources, primarily under soluble nutrient-depleted conditions.

Glutaminolysis drives membrane trafficking to promote invasiveness of breast cancer cells.

The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness.

Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion.

The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma-whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis-indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo.

Extracellular matrix endocytosis in controlling matrix turnover and beyond: emerging roles in cancer.

The extracellular matrix (ECM) is a network of secreted proteins that, beyond providing support for tissues and organs, is involved in the regulation of a variety of cell functions, including cell proliferation, polarity, migration and oncogenic transformation. ECM homeostasis is maintained through a tightly controlled balance between synthesis, deposition and degradation. While the role of metalloproteases in ECM degradation is widely recognised, the contribution of ECM internalisation and intracellular degradation to ECM maintenance has been mostly overlooked. In this review, I will summarise what is known about the molecular mechanisms mediating ECM endocytosis and how this process impacts on diseases, such as fibrosis and cancer.

Endosomal integrin signals for survival.

The mechanisms underlying integrin-dependent signalling are a topic of continued study. Endocytosed integrins are now shown to drive assembly of signalling complexes on the cytoplasmic face of endocytic membranes to promote cancer cell survival and increase metastatic capacity following cell detachment.

Ligand-Occupied Integrin Internalization Links Nutrient Signaling to Invasive Migration.

Integrin trafficking is key to cell migration, but little is known about the spatiotemporal organization of integrin endocytosis. Here, we show that α5ß1 integrin undergoes tensin-dependent centripetal movement from the cell periphery to populate adhesions located under the nucleus. From here, ligand-engaged α5ß1 integrins are internalized under control of the Arf subfamily GTPase, Arf4, and are trafficked to nearby late endosomes/lysosomes. Suppression of centripetal movement or Arf4-dependent endocytosis disrupts flow of ligand-bound integrins to late endosomes/lysosomes and their degradation within this compartment. Arf4-dependent integrin internalization is required for proper lysosome positioning and for recruitment and activation of mTOR at this cellular subcompartment. Furthermore, nutrient depletion promotes subnuclear accumulation and endocytosis of ligand-engaged α5ß1 integrins via inhibition of mTORC1. This two-way regulatory interaction between mTORC1 and integrin trafficking in combination with data describing a role for tensin in invasive cell migration indicate interesting links between nutrient signaling and metastasis.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

The diacylglycerol kinase α/atypical PKC/ß1 integrin pathway in SDF-1α mammary carcinoma invasiveness.

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5ß1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of ß1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and ß1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - ß1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.

Late endosomal and lysosomal trafficking during integrin-mediated cell migration and invasion: cell matrix receptors are trafficked through the late endosomal pathway in a way that dictates how cells migrate.

Recently it has become clear that trafficking of integrins to late endosomes is key to the regulation of integrin expression and function during cell migration. Here we discuss the molecular machinery that dictates whether integrins are sorted to recycling endosomes or are targeted to late endosomes and lysosomes. Integrins and other receptors that are sorted to late endosomes are not necessarily degraded and, under certain circumstances, can be spared destruction and returned to the cell surface to drive cell migration and invasion. We will discuss how the exchange of adhesion receptors and other key regulators of cell migration between late endosomes/lysosomes and the plasma membrane can promote dynamic turnover of adhesions during cell migration.

Neuropilin-1-dependent regulation of EGF-receptor signaling.

Neuropilin-1 (NRP1) is a coreceptor for multiple extracellular ligands. NRP1 is widely expressed in cancer cells and in advanced human tumors; however, its functional relevance and signaling mechanisms are unclear. Here, we show that NRP1 expression controls viability and proliferation of different cancer cells, independent of its short intracellular tail. We found that the extracellular domain of NRP1 interacts with the EGF receptor (EGFR) and promotes its signaling cascade elicited upon EGF or TGF-α stimulation. Upon NRP1 silencing, the ability of ligand-bound EGFR to cluster on the cell surface, internalize, and activate the downstream AKT pathway is severely impaired. EGFR is frequently activated in human tumors due to overexpression, mutation, or sustained autocrine/paracrine stimulation. Here we show that NRP1-blocking antibodies and NRP1 silencing can counteract ligand-induced EGFR activation in cancer cells. Thus our findings unveil a novel molecular mechanism by which NRP1 can control EGFR signaling and tumor growth.

PKD controls αvß3 integrin recycling and tumor cell invasive migration through its substrate Rabaptin-5.

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvß3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvß3, which in turn inhibits α5ß1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvß3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvß3-dependent invasion. Conversely, invasive migration that is dependent on α5ß1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration.

Inhibition of αvß3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5ß1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5ß1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvß3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvß3 to RCP and for this Rab11 effector to drive the trafficking of α5ß1 that is required for tumor cell invasion through three-dimensional matrices.

SAP-mediated inhibition of diacylglycerol kinase α regulates TCR-induced diacylglycerol signaling.

Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.