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6.1 Introduction

Although endocytosis has long been regarded as a conduit for the internalization and degradation of nutrients and cell surface receptors, it is now accepted that the endosomal membrane system plays a vital role in the control and execution of ­spatially restricted functions, such as cell adhesion and motility. Cell adhesion is essential for the maintenance of multicellularity in living organisms. Intercellular adhesion and cell-to-extracellular matrix (ECM) adhesion are a result of the assembly and functional integrity of “adhesion complexes” at sites of cell–cell or cell–ECM contacts, respectively [34]. These complexes consist of transmembrane adhesion molecules coupled to intracellular scaffold or signaling proteins and the cytoskeleton. Cadherin family cell adhesion molecules and their associated scaffold proteins, the catenins, are major components of cell–cell adhesive contacts, namely, the adherens junctions and desmosomes [34]. The major transmembrane protein at cell–ECM adhesive contacts (i.e., focal adhesions and hemidesmosomes) is the heterodimeric integrin receptors [34]. These cell–cell and cell–ECM adhesion complexes are linked to and stabilized by the actin or intermediate filaments but undergo significant remodeling during the acquisition of migratory and invasive phenotypes in tumor cells [93, 94]. In the initial stages of tumor progression, tumor cells sometimes undergo epithelial–mesenchymal transition, which requires the disruption of cell–cell adhesions [5]. The trafficking of cadherins along the endocytic pathway is now accepted as an important mechanism involved in the remodeling of cell–cell adhesions [39]. Some cancer cells maintain or reestablish cell–cell adhesions during metastasis and their collective migration requires that both cell–cell and cell–ECM adhesions stay intact [25]. Mesenchymal tumor cells will migrate and invade into the basement membrane and surrounding stromal tissues [54]. To facilitate migration and invasion, tumor cells continuously form new cell–ECM adhesions at the leading edge, whereas focal adhesions at the trailing edge are disrupted by endocytosis.

Besides serving as carriers and sorting stations for the trafficking of adhesion molecules, emerging evidence suggests that endosomal compartments are also essential sites of signal transduction [53, 77, 84]. Activated receptors can function in endosomes, and certain signaling components are localized, even exclusively, to endosomes. Signals transmitted from endosomes are robust and typically long-­lived, different from those that arise from the plasma membrane. Endosomal signaling is widespread across species and regulates essential processes including growth and differentiation in addition to cell adhesion and motility. Subversion of the mechanisms involved is predicted to play an important role in several human diseases and, most especially, cancer. Pharmacological agents that target receptor signaling at the plasma membrane have proved to be effective therapeutics for some cancers [53]. Thus, selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways that are altered in cancers, could also provide novel therapies for tumor progression.

In this review chapter, we discuss current evidence coupling endocytosis and the regulation of signaling pathways in cells and how altered regulation of these pathways can lead to acquisition of oncogenic phenotypes. We also describe how endocytosis and recycling of adhesion molecules, such as cadherins and integrins, is coming into focus as a major mechanism in the regulation of adhesive and migratory properties of cells.

6.2 Endocytosis Regulates Signal Transduction

Endocytosis plays an important role in the duration and extent of signal transduction via controlling the number and accessibility of cell surface receptors [52, 82, 84]. Signal transduction starts at the cell surface when ligands such as growth factors and hormones bind to their cognate receptors, which recruit and activate signaling molecules such as adaptors and enzymes (e.g., kinases) that further activate downstream effectors to amplify the signal transduction processes ultimately leading to regulation of gene expression, cell proliferation, or cell differentiation. Malfunction of the signal transduction processes is a major cause of cancer.

Receptor tyrosine kinases (RTKs) constitute a large family of receptors whose signal transduction processes promote cell proliferation or differentiation. A well-­characterized example is the epidermal growth factor receptor (EGFR). Ligand (EGF) binding causes conformational changes, dimerization, and tyrosine phosphorylation of EGFR, which activates phosphoinositide 3-kinase (PI3-K), phospholipase Cγ (PLCγ), and the mitogen-activated protein (MAP) kinase pathways at the cell surface. The ligand also induces endocytosis of EGFR into endosomes and eventually intraluminal vesicles (ILVs) and lysosomes for degradation, which reduces the number of active EGFR molecules and attenuates the signaling in the so-called “downregulation” process. This process is delayed under hypoxia conditions characteristic of cancer cells where hypoxia-inducible factor HIF1α blocks Rabaptin-5 expression, leading to reduced Rab5-mediated endosome fusion and endocytosis [91]. As a result, EGFR signaling is prolonged with increased cell proliferation.

EGFR in endosomes, before delivery to ILVs and lysosomes, may remain engaged to the ligand, and its cytoplasmic domain may continue signaling. Indeed, the first evidence of endosomes serving as a platform for EGFR signaling inside the cell came from a study that showed reduced phosphorylation/activation of MAP kinases in the cells where endocytosis was blocked by a dynamin mutant [90]. This concept was later generalized as the signaling endosome hypothesis [32]. Signaling endosomes not only continue the signaling processes initiated at the cell surface but also gain access to new signaling molecules and start new signaling processes. For example, EGFR can activate the small GTPase Rab5 via RIN1 on endosomes [85]. Rab5-GTP and certain receptors can directly recruit APPL1 to endosomes, which activates Akt and regulates its substrate specificity for GSK-3β, a process critical for cell survival and embryonic development in zebrafish and possibly other vertebrates [76]. On the other hand, an intriguing recent observation suggests that accumulation of activated EGFR on endosomes via blocking ILV formation triggers apoptosis [72], although the signaling pathway is yet to be established. It is clear that the endosomes provide a membrane environment distinct from that of the plasma membrane and their mobility inside cells allows access to additional signaling molecules leading to new functional consequences.

Endosomal transport is critical for retrograde signaling by nerve growth factor (NGF) and its receptor (TrkA) in neuronal survival, migration, axon growth, and target cell innervation [3, 10]. In this case, target-derived NGF binding at the tip of the axon induces endocytosis of the NGF–TrkA complex into the signaling endosomes that start signaling locally inside the axon and undergo retrograde transport along microtubules towards the cell body/soma. The signaling endosomes carry signaling molecules such as the activated TrkA, ERKs, and Akt [28, 32] as well as specific transcription factors (e.g., CREB) translated in the axon [11] to activate specific nuclear gene expression in the soma, including a positive feedback loop of increased expression of TrkA [48]. The functional consequences are the aforementioned neurotrophic effects.

Endosomal membrane is enriched with PI3P, which recruits PI3P-binding signaling molecules such as the FYVE domain-containing proteins important for TGFβ receptor- and G protein-coupled receptor (GPCR)-mediated signal transduction processes. In the case of TGFβ signaling, the TGFβ receptor is endocytosed into endosomes where it interacts with SMAD anchor for receptor activation (SARA), a FYVE domain-containing adaptor protein that recruits SMAD2 to the endosomes for phosphorylation by the TGFβ receptor [88]. The phosphorylated SMAD2 then forms a complex with SMAD4, followed by translocation to the nucleus for activation of gene expression. In the case of GPCR signaling, activated G protein on endosomes can activate the PI3K Vps34 to produce PI3P that in turn recruits FYVE domain-containing proteins to promote MAPK and Cdc42 signaling pathways [84].

Endocytosis also plays an important role in Notch-mediated signal transduction and neuronal cell proliferation [22, 39]. Both Notch and its ligands (the Delta/Serrate/Lag2 (Dsl) domain-containing proteins) are transmembrane proteins on apposing cells. Ligand binding leads to two consecutive proteolytic cleavages (S1 and S2) in the ectodomain of Notch. The C-terminal fragment of Notch is then endocytosed, followed by another cleavage (S3) in the transmembrane domain by γ secretase to release the cytoplasmic intracellular domain that translocates to the nucleus and activates expression of target genes. Interestingly, the ligand itself, e.g., Delta, requires endocytosis and recycling to concentrate at specific regions on the plasma membrane for efficient binding and activation of Notch on the signal-­receiving cells [22, 39].

An important mechanism that controls the endocytosis and/or endosomal sorting of the signaling receptors involves ubiquitination at their cytoplasmic domains. While polyubiquitination with the Lys-48 linkage marks the substrate for degradation by the proteosome, polyubiquitination with the Lys-63 linkage and monoubiquitination may regulate other functions of the substrate including endocytosis and endosomal sorting [84]. Ubiquitination is necessary for the endocytosis of Ste2, a GPCR, in yeast [29], but it is not essential for other GPCRs and RTKs in animal cells [20, 79]. In the latter case, ubiquitination may still increase the interaction of these signaling receptors with components of clathrin-coated pits to facilitate their endocytosis [2, 36]. Importantly, ubiquitination is critical for subsequent sorting of signaling receptors into ILVs/MVBs [20, 79], via interaction with the ESCRT complexes, to terminate the signal [35, 81, 82]. Consistently, ubiquitination-deficient EGFR shows increased signaling activity. A well-documented E3 ubiquitin ligase, Cbl, is responsible for the ubiquitination of various RTKs. Mutations that abrogate Cbl ubiquitin ligase activity are known to cause cancer such as myeloid leukemia and lung cancer [74, 86], suggesting that ubiquitination-mediated RTK sequestration in ILVs/MVBs and signal termination are critical for normal signal transduction processes and cell growth. In the case of Notch, non-ligand-bound Notch is ubiquitinated by the E3 ligase ITCH, endocytosed and delivered to lysosomes for degradation [8].

In addition to signaling receptors, ubiquitination of downstream signaling molecules on the plasma membrane, such as the Ras GTPases (H- and N-Ras), leads to endocytosis and signal downregulation in both mammalian cells and Drosophila where the system controls organ development [96, 97]. The E3 ubiquitin ligase responsible for Ras ubiquitination is Rabex-5 [96, 97], which was originally identified as a GEF for activation of the endosome-associated Rab5 and endosome fusion [31]. Rabex-5 targets early endosomes and plasma membrane by binding to Rab22 [99, 100] or by forming a complex with Rabaptin-5 that in turn binds to Rab5 [31]. The K-Ras isoform, on the other hand, is not ubiquitinated, but a recent study shows that a fraction of K-Ras is endocytosed via the clathrin-dependent pathway and follows the conventional endocytic pathway to early endosomes, late endosomes/MVBs, and lysosomes [46]. Interestingly, K-Ras is able to recruit Raf1 to elicit signal transduction on late endosomes [46], which normally degrade endocytosed cargoes and reduce signaling.

Endocytosed receptors are not always destined to degradation in late endosomes and lysosomes; instead they can be recycled to the plasma membrane for reutilization and additional rounds of signaling, depending on the types of ligands and endocytic portals. For example, TGFα also interacts with EGFR but its affinity is lower than EGF and readily dissociates from the receptor in early endosomes [19, 24]. As a result, the receptor is sorted back to the plasma membrane, due to insufficient ubiquitination [45], which likely contributes to the high potency of TGFα in promoting tumor cell growth [56]. In addition, receptors can be endocytosed via clathrin-­dependent or clathrin-independent pathways or both [17, 81]. In the case of EGFR, low concentrations of EGF induce clathrin-dependent endocytosis while high concentrations of EGF promote clathrin-independent endocytosis [81]. The former pathway largely recycles EGFR back to the plasma membrane for continual signaling and the latter pathway promotes EGFR traffic to late endosomes and lysosomes for degradation and signal attenuation [81]. In addition to the RTKs, some GPCRs such as the β2 adrenergic receptor (β2AR) require endocytosis and recycling to be resensitized for sustained signaling. In this case, β2AR can be inactivated by phosphorylation at the plasma membrane and endocytosis allows β2AR to gain access to the endosome-associated phosphatase 2A for dephosphorylation and resensitization [66]. The active β2AR is then recycled back to the plasma membrane, via a Rab4-dependent fast recycling pathway, for ligand binding and new rounds of signaling [21, 59, 78].

6.3 EMT in Cancer

Most solid tumors are epithelial in origin. A loss of epithelial cell markers and concomitant acquisition of mesenchymal cell markers have been observed in some epithelial tumors, such as non-small cell lung cancer (NSCLC) and pancreatic, colorectal, and hepatocellular cancers particularly at the invasive front [37, 87]. This profound phenotypic conversion, referred to as epithelial to mesenchymal transition (EMT), is orchestrated by integrated networks of signal transduction pathways that direct marked alterations in cell adhesion and motility. Although EMT is best known for its role in embryonic development, in the adult, several oncogenic pathways (growth factors, Src, Ras, Wnt/beta-catenin, and Notch) may induce EMT [87].

Although there are accumulating data to suggest a critical role for EMT in cancer progression, the demonstration of this process in human cancer has been controversial [67]. Moreover, the acquisition of mesenchymal phenotypes is not a prerequisite for cell migration/invasion and in many cases appears to be characteristic of only a few cells at the invasive fronts in a tumor mass. The evidence to date suggests that the induction of EMT depends on the tumor type and its genetic alterations as well as on its interaction with the extracellular matrix [67]. Over the last decade, however, it’s been shown that some cancer cells reactivate EMT in an effort to escape their normal boundaries [67]. The loss of epithelial cell markers (e.g., E-cadherin) is associated with disease progression and metastatic potential of a tumor. There is accruing evidence that cancer cells can dedifferentiate through activation of specific biological pathways associated with EMT, thereby gaining the ability to migrate and invade. Hence, what has been observed experimentally regarding EMT and normal embryonic development is also thought to apply in the progression of solid tumors—a cellular reprogramming process whereby epithelial tumor cells lose cell polarity and cell junction proteins and at the same time gain signal transduction activities associated with cell invasion and survival in an anchorage-­independent environment. Mesenchymal-like tumor cells gain migratory capacity at the expense of proliferative potential. Cellular changes resulting in EMT in cancer are thought to play a major role in disease progression and have been associated with poor prognosis in patients [37].

6.4 Endocytic Trafficking of Cadherins in EMT

A critical molecular event underpinning the dissolution of cell–cell contacts during EMT is the loss of the cell–cell adhesion molecule, E-cadherin, a key component of the adherens junctions [87]. EMT and metastatic progression are most often associated with a reversible downregulation of E-cadherin (encoded by CDH1) involving either hypermethylation of the CDH1 promoter or repression by EMT-inducing transcription factors [5, 87]. In particular, EMT is accompanied by the activation of two related zinc finger-containing transcription factors, Snail and Slug. The basic helix–loop–helix proteins, Twist 1 and Twist 2, and the ZEB family proteins have also been shown to induce EMT via transcription regulation. Notably, however, in addition to transcriptional downregulation, posttranscriptional regulation of adhesive structures can also markedly influence the progression of EMT [12]. The endocytosis and lysosomal degradation of E-cadherin as described below is one such cellular mechanism that can have a profound impact on the initial stages of EMT.

The cytoplasmic domain of E-cadherin contains a dileucine motif, which is a binding site for clathrin adaptor complexes, and mutations in the motif inhibit E-cadherin endocytosis [50]. The E-cadherin dileucine motif also binds to p120ctn, which in polarized poorly motile epithelia masks the dileucine motif to prevent the endocytosis of E-cadherin [51]. Cellular depletion of p120ctn results in the internalization of cadherins and loss of cell–cell contacts [9, 14, 95]. The binding of an adaptor molecule, Numb, to p120ctn negates the p120ctn-mediated suppression of E-cadherin endocytosis since Numb recruits the AP-2 clathrin adaptor complex promoting endocytosis [75]. Numb can also interact with the NVYYY motif on E-cadherin, which in turn can hinder the p120ctn–E-cadherin interaction [92].

Growth factors such as HGF, as well as oncogenic v-Src, can also induce epithelial–mesenchymal transition (EMT) in part by promoting the endocytosis of cadherin molecules [12, 94]. Src-mediated phosphorylation of the NVYYY motif on E-cadherin induces the dissociation of p120ctn and recruits a c-Cbl-related E3 ubiquitin ligase, Hakai [26]. Hakai induces the ubiquitination of E-cadherin and subsequently its endocytosis and lysosomal degradation, which require the activation of the Rab small GTP-binding proteins, Rab5 and Rab7 [63]. HGF treatment or v-Src expression activates ARF6, an ARF family small GTP-binding protein, which enhances E-cadherin endocytosis [60]. In Madin–Darby Canine Kidney (MDCK) cells, ARF6-GTP recruits the nucleoside diphosphate kinase, Nm23-H1, initiating a downregulation of Rac1 activity and promoting the clathrin-dependent endocytosis of E-cadherin to the early endosome, both of which facilitate the disassembly of the adherens junctions [62]. Furthermore, upon Src-induced loss of cell–cell contacts, internalized E-cadherin is targeted to the lysosome for degradation in an ARF6-­dependent manner so that it cannot be recycled to the plasma membrane, thus ensuring that cell–cell contacts will not be reformed. The TBC/RabGAP Armus is thought to play a role in this process, bridging signaling between ARF6, Rac1, and Rab7 [23]. Armus was shown to bind Rac1 and locally facilitate lysosome biogenesis and the degradation of E-cadherin. Inhibition of ARF6 activity, by expression of a dominantly interfering mutant, ARF6(T27N), enhances the epithelial phenotype by preventing the internalization of E-cadherin into endosomal compartments and thereby blocking hepatocyte growth factor (HGF) and Src-induced cell scattering [60, 61]. In addition, the downregulation of ARF6 activity, established by a positive feedback loop between EphA2 and E-cadherin, has been shown to enhance E-cadherin-based adhesion and the maturation of apical–basal polarity in MDCK cells [49]. The formation of stable adherens junctions during epithelial cell polarization is dependent on the spatially regulated activation of ARF6, which is modulated by the formation of a complex between FRMD4A/GRSP-1/PAR3 and the ARF6-GEF cytohesin-1 at cell junctions [33]. HGF also induces Ras-mediated activation of RIN2, an activator for Rab5, which also facilitates the endocytosis of E-cadherin [38].

The aforementioned effects of ARF6 on the internalization of E-cadherin and other cell surface receptors have also been shown to impact epithelial glandular organization in 3D cell cultures [89]. In this regard, sustained ARF6 activation in basement membrane cultures of epithelial cysts, a structural unit of epithelial glandular organs, leads to the internalization of E-cadherin as well as growth factor receptors by ARF6-regulated pathways. Sustained signaling from endosomes (described further below), in turn, leads to the formation of aberrant glandular morphologies, reminiscent of tumorigenic phenotypes seen in vivo [16].

In addition to clathrin-mediated endocytosis, caveolin-mediated endocytosis is also involved in E-cadherin internalization in some cell types [1, 47]. EGF treatment of MCF-7 cells, on the other hand, promotes E-cadherin into endosomes via clathrin-­independent pathways [64]. While multiple internalization routes have been implicated in E-cadherin endocytosis, collectively these findings show that the cycling of E-cadherin along the endosomal pathway can markedly impinge on the dynamics of the adherens junctions of epithelial tissues and loss of the epithelial phenotype.

6.5 Endocytic Trafficking of Integrins and Acquisition of Motile Phenotypes

Migrating and invading cells display an increased internalization and recycling of integrins from the retracting edge of the cell to the leading edge and at sites of cell invasion. β1 integrin receptors have been localized to clathrin-coated pits [15] where the endocytic adaptors Numb and Dab2 bind to integrin receptors to facilitate internalization [57]. HS1-associated protein X-1 (HAX-1) has been shown to bind to the cytoplasmic tail of β6 integrin to facilitate the clathrin-mediated internalization of αvβ6 integrin receptors, which in turn enhances migration and invasion of oral sqaumous carcinoma cell lines [69]. While the above is one example, integrin trafficking has been associated with migratory potential of several tumor cells lines [54, 70]. Integrin endocytosis is also thought to facilitate the uptake of ECM proteins such as fibronectin and vitronectin and transport of these molecules to the lysosomes [43, 80].

In actively migrating cells, ARF6 and Rab family GTPases have been linked to the trafficking of integrin receptors. In this regard, β1 integrin has been shown to localize to ARF6-regulated recycling endosomes [6, 68]. Expression of dominant-­negative ARF6 inhibits the cell’s ability to recycle this endosomal compartment to the plasma membrane leading to a decrease in receptors at the migrating edge [6, 68]. In addition, the recycling of β1 integrin is regulated by an ARF6-GAP, ACAP1; the inhibition of ACAP1 or its phosphorylation by Akt inhibits integrin recycling and cell migration [41]. The ARF6 GEF, GEP100, which is upregulated in breast cancers, has also been implicated in the trafficking of β1 integrin receptors [18, 73]. The migratory ability of cells is also modulated by engagement with the extracellular matrix and requires ARF6-mediated activation of Rac1 for the formation of protrusive structures such as lamellipodia at the leading edge [13]. The formation of an α4 integrin–paxillin–Arf-GAP complex at the trailing edge of the cell assists in directional migration by inhibiting ARF6 activity, thereby blocking adhesion-­dependent Rac1 activation and the extension of lamellipodia [58]. Slit2-Robo signaling can block the ARF6 and Rac1 activation induced by integrin engagement, important in pathologies of endothelial cell protrusive activity [44].

A subset of Rab GTPases on the plasma membrane, early and recycling endosomes control the integrin trafficking, and altered expression levels of these Rabs are often associated with various types of cancer [40]. Rab5 and Rab21 are associated with the plasma membrane and early endosomes and regulate the internalization of β1 integrins via direct interaction with their α subunits [65]. Reduction of Rab5 or Rab21 in carcinoma-associated fibroblasts can decrease α5 integrin at the plasma membrane and remodeling of cell–extracellular matrix interaction, which is required for the invasion of squamous cell carcinoma [30]. Indeed, Rab5 is highly expressed in lung adenocarcinoma and hepatocellular carcinoma [27, 42]. The Rab11 subfamily members (Rab11 and Rab25) are localized in a perinuclear recycling compartment that controls a slow recycling pathway important for trafficking of integrins back to the plasma membrane at the leading edge. Rab11 appears to collaborate with ARF6 to control the exit of integrins from the perinuclear recycling compartment [68]. Importantly, Rab11 controls α6β4 integrin recycling involved in hypoxia-induced breast cancer cell invasion [98]. Rab25 is related to Rab11 and directly binds to the β1 subunit of α5β1 integrin to facilitate its recycling to the leading edge of the plasma membrane, a process implicated in cancer cell invasion and metastasis [7]. Overexpression of Rab25 is well documented in many types of cancers [40] including ovarian cancer, breast cancer, testicular tumor, Wilms tumor, and bladder and hepatocellular carcinomas. Interestingly, Rab25 expression is decreased in colorectal adenocarcinomas, which correlates with poor prognosis of colon cancer patients [55]. In this regard, mouse models of colon cancer show that loss of Rab25 can increase colonic tumor formation [55]. It is not yet clear why different types of cancers require overexpression and loss of Rab25, respectively, to promote cell invasion and aggressiveness. In contrast to Rab11/Rab25-mediated slow recycling, Rab4 is localized in a fast recycling compartment and facilitates the fast recycling of certain integrins (e.g., αvβ3) to the cell surface in response to the platelet-derived growth factor (PDGF) [71].

6.6 Concluding Remarks

Endocytosis has emerged as an important regulatory mechanism in signal transduction/cell proliferation, cell polarity/EMT, and cell adhesion/invasion, in addition to its conventional role in the uptake and digestion of nutrients. The regulatory ­function of endocytosis in these processes involves internalization and sorting of signaling receptors, cell junction molecules (e.g., cadherins), and integrins to degradative or recycling pathway. Aberrant endocytic machinery and unbalanced endocytosis can contribute to uncontrolled cell proliferation, EMT, and aggressive cell invasion, which are hallmarks of cancer cells. Indeed, altered expression and/or mutations in endocytic genes are frequently found in tumors [82]. One well-documented example is Cbl, an E3 ubiquitin ligase involved in ubiquitination and endocytosis of signaling receptors. Inactivating mutations in Cbl are associated with myeloid malignancies [74]. In addition, mutations in the cytoplasmic domain of signaling receptors may abrogate their endocytosis and reduce degradation, contributing to tumorigenesis. For example, ErbB-2, an endocytosis-defective variant of EGFR, shows strong transforming effect and is highly expressed in breast cancer [4, 83]. A better understanding of the endocytic machinery that regulates the degradation and recycling of signaling receptors, cadherins, and integrins should help develop novel and specific therapeutics against cancer.