Dynamics of receptor trafficking in tumorigenicity

Review

Dynamics of receptor trafficking in tumorigenicity Christine A. Parachoniak1,2 and Morag Park1,2,3,4 1

Department of Biochemistry, McGill University, Montreal, Quebec, Canada Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada 3 Department of Medicine, McGill University, Montreal, Quebec, Canada 4 Department of Oncology, McGill University, Montreal, Quebec, Canada 2

The transport and sorting of cell surface receptors into membrane-bound intracellular compartments is crucial for cellular homeostasis. Defects in receptor trafficking are associated with several diseases, including cancer. Recent advances in our understanding of mechanisms that control receptor trafficking have highlighted the involvement of membrane trafficking in cell signaling, as well as in biological processes, including cell migration and invasion. In this review, we summarize current knowledge of how cargos, focusing on receptor tyrosine kinases (RTKs) and integrins, are dynamically transported through the endosomal pathway for recycling, and how this promotes spatially restricted signaling microdomains associated with distinct biological responses. We discuss mechanisms through which dysregulation of membrane trafficking contributes to tumorigenesis and potential therapeutic approaches. Trafficking from the plasma membrane Cells respond to signals from the extracellular environment through the activation of intracellular signaling programs that lead to a biological response. For this, plasma membrane receptors recognize and bind to external signals and transmit them into intracellular signaling cascades. Therefore, the ability of cells to evoke a biological response is dependent, in part, on the availability of receptors present at the cell surface. Growth factor receptors constitute one example of transmembrane proteins that serve such a purpose, with the family of RTKs one of the most prominent subgroups. Upon ligand binding, RTKs become catalytically active and tyrosine phosphorylated, enabling the recruitment of signaling proteins to initiate downstream signaling cascades. This process is balanced by the simultaneous recruitment of endocytic adaptor proteins, which enhance internalization of RTKs into the endocytic trafficking network, thereby allowing for their removal from the cell surface. In addition to regulating signal termination, it is now recognized that internalization and entry into endocytic compartments is an integral part of signaling, controlling the strength, and spatial and temporal restrictions to RTK signals [1,2]. In the past few years, defects in pathways Corresponding author: Park, M. ([email protected]). Keywords: endocytosis; receptors; cancer; recycling; integrin; receptor tyrosine kinase

that regulate endocytosis and recycling have emerged as underlying mechanisms contributing to cancer. In this review we focus on molecular mechanisms that modulate intracellular trafficking and recycling and how this influences signaling outcomes, particularly as it relates to tumorigenesis. Entry, sorting and transport of cargo RTKs, once internalized, enter early endosomes and are sorted towards one of two fates: to late endosomes and lysosomes for degradation or recycled (directly or indirectly) back to the plasma membrane (Figure 1). To date, clathrin-dependent endocytosis (CDE) remains the best characterized internalization pathway; however, alternative clathrin-independent endocytosis (CIE) pathways, such as caveolin-dependent, macropinocytosis or phagocytosis pathways, provide alternate modes of cargo entry [3]. In the case of the epidermal growth factor receptor (EGFR), several studies support CDE as the major entry portal for EGFR under conditions of low, physiological concentrations of EGF [4–7], with roles for other pathways being reported under specific ligand and/or cell-type conditions [6,8,9]. In CDE, clathrin triskelions interact with multiple endocytic adaptor proteins, some of which are cargo specific, to promote the capture of cargo into inward budding vesicles. Four distinct mechanisms, including receptor ubiquitination, recruitment of adaptor proteins AP-2 and Grb2 and receptor acetylation, cooperate in internalization of the EGFR, highlighting the plasticity of the endocytic system [10]. Nascent vesicles subsequently pinch off from the plasma membrane with the aid of the GTPase dynamin, rapidly shed their clathrin coat and fuse with the early endosome [11]. Like RTKs, integrins are heterodimeric transmembrane proteins that are also internalized through CDE and CIE pathways [12]. In the case of CDE, the cytoplasmic tail of the integrin beta-subunit contains NXXY and NPXY motifs that serve to directly recruit endocytic adaptors [12,13]. Alternatively, certain integrins, including avb3 and a5b1, have been localized to caveolae, suggesting that some integrins can use CIE for internalization [12]. However, the mechanism(s) responsible for recruitment of integrins to these alternative pathways remain ill defined. Early endosomes are enriched for phosphatidylinositol-3phosphate (PtdIns(3)P) either through the action of the class III PI3K, VPS34 or dephosphorylation of PtdIns(3,4,5)P3 by

0962-8924/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2012.02.002 Trends in Cell Biology, May 2012, Vol. 22, No. 5

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P

CCP

P

P

EE

Degradation

Recycling ERC

Key:

MVB

Receptor

Adaptors Dynamin Rab5

Rab4

ESCRTs Rab11

P

phosphotyrosine Hsp70

Rab7

Clathrin ligand

TRENDS in Cell Biology

Figure 1. Receptor-mediated endocytosis. Upon ligand binding, receptors are rapidly cleared from the plasma membrane with the aid of endocytic adaptor proteins, which promote inclusion into clathrin-coated pits (CCPs). Newly formed clathrin-coated vesicles rapidly shed their coat and fuse with early endosomes (EEs), where they are sorted towards either a recycling or a degradative fate. Ubiquitinated receptors are recognized by the endosomal sorting complex required for transport (ESCRT) machinery and sorted into multivesicular bodies (MVBs) that subsequently fuse with lysosomes and are degraded. Alternatively, some receptors recycle directly up to the plasma membrane or enter an endocytic recycling compartment (ERC) before returning to the cell surface.

a cascade including PI 5- and PI 4-phosphatases [14]. PI3P, together with the small GTPase Rab5, organizes the early endosome into distinct subdomains to coordinate the recruitment of complexes, such as early endosome antigen 1 (EEA-1) and sorting nexins (SNX), through their PtdIns(3)P binding domains, the FYVE finger or Phox-homology (PX) domains, respectively [14]. The early endosome accepts incoming endosomes for a brief period of time before being transported along microtubules and maturing into a late endosome [15]. During this time, cargo at the early endosome may be quickly recycled back up to the plasma membrane through tubular extensions of the early endosome, transported to the endocytic recycling compartment (ERC), or it may progress to the late endosomal compartment. Endosomes transitioning from early to late endosomal compartments are accompanied by the gradual loss of Rab5 and the acquisition of Rab7. From studies in Caenorhabditis elegans, the protein SAND-1/Mon1 controls this conversion. SAND-1/Mon1 simultaneously coordinates inhibition of Rab5 activation, through delocalization of the 232

Rab5 guanine exchange factor (GEF), Rabex-5, and activation of Rab7 through the recruitment of the Rab7 GEF, HOPS complex [16]. Concomitant with endosome maturation, cargos that are ubiquitinated are captured into flat clathrin-bilayer regions of endosomes [17] by four endosomal sorting complex required for transport (ESCRT) multiprotein complexes, namely ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, and associated accessory proteins [18,19]. From here, the limiting membrane of the late endosome can invaginate and bud into the lumen to form a multivesicular body (MVB) [18]. These structures subsequently fuse with lysosomes promoting cargo degradation [20]. This is a crucial step in RTK downregulation because, once sequestered into an intraluminal vesicle (ILV), the signaling competent C-terminus of the receptor is occluded from cytosolic signal transduction proteins. Receptor signaling from endosomes Purification of the endosomal compartment from liver parenchyma following stimulation with EGF revealed that

Review components of the MAPK signaling cascade including Shc, Grb2 and SOS as well as the downstream target, Raf, are activated and localize to endosomes [21]. This landmark study provided support that signaling can occur from regions other than the plasma membrane, and is supported by later fluorescence resonance energy transfer (FRET) studies that detected EGFR–Grb2 complexes in endosomal compartments [22]. Further evidence was provided by studies using cells defective in dynamin-mediated endocytosis by expressing a catalytically inactive dynamin mutant (K44A) [23]. This severely impaired EGF-dependent endocytosis of the EGFR and suppressed phosphorylation of a subset of proteins that lie downstream of EGF, including ERK1, ERK2 and the p85 subunit of PI3-kinase [23]. The ability to maintain sufficient receptor signaling to elicit a biological response after rapid removal from the plasma membrane may be particularly relevant in vivo, where ligand concentration can be limited [1]. Consistent with this model, upon EGF stimulation, the MAPK scaffold complex containing MEK1 partner 1 (MP1) and p14 localizes to late endosomes and is required for full activation of ERK1/ERK2 signaling during embryonic development [24], whereas the Rab5 effector Appl1 regulates Akt activity from endosomes to mediate cell survival during zebrafish development [25]. Similarly, overexpression of the small GTPase Rab5 impairs directional migration of primordial germ cells to gonads in zebrafish [26]. In cell culture, knockdown of subunits of the ESCRT-0 complex, hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) or ESCRT-I complex, tumor susceptibility gene 101 (Tsg101), attenuate entry of Met, the RTK for HGF or EGFR into MVBs, resulting in prolonged receptor stability and sustained ERK1/2 activation [27,28]. Thus, it is generally accepted that endosomes are unique signaling platforms on which to assemble RTK signaling complexes [1,2]. Mechanisms and fate of receptor entry into recycling pathways Although still mechanistically ill defined, recycling back to the plasma membrane can occur rapidly and directly from the early endosome through a Rab4-dependent pathway, more slowly through an intermediate Rab11-dependent endocytic recycling compartment (ERC), or through alternative Rab- and/or Arf-mediated pathways [15,29]. Fast recycling has largely been ascribed to bulk recycling; the high surface-to-volume ratio of tubular extensions of the early endosome pinch off in a sequence-independent manner, typified by the recycling of the transferrin receptor in the presence of its ligand, diferric transferrin [15]. Recently, positive-selection mechanisms for distinct receptor recycling pathways are beginning to emerge. In the case of the Met RTK, HGF ligand activation can promote entry of the receptor to an early Rab4-dependent recycling pathway [30]. This localization to a Rab4 compartment, and subsequent recycling back to the plasma membrane, requires recruitment of the monomeric adaptor protein, Golgi-localized, gamma ear-containing, ARF-binding protein 3 (GGA3) to the Met receptor signaling complex. Upon HGF stimulation, activation of the small GTP-binding protein Arf6 and recruitment of the signaling adaptor Crk cooperate in the recruitment of GGA3 to Met-positive

Trends in Cell Biology May 2012, Vol. 22, No. 5

endosomal membranes. Following GGA3 knockdown, Met is no longer retained within the Rab4 recycling compartment and efficiently enters a degradative compartment [30]. Although three GGA family members are found in humans, only GGA3 engages with Met. This specificity is dependent on the ability of GGA3 to form a complex with Crk, for which GGA3, but not GGA1 or GGA2, contains proline-rich sequences within its hinge region that interact with the Crk SH3 domain. Interestingly, GGA1 has also been observed on rapid recycling structures [31], suggesting that, similar to GGA3, GGA1 may also promote recycling of an as yet unspecified cargo. Entry of cargo into recycling endosomes is associated with signaling. Diversion of Met from the recycling pathway, following GGA3 knockdown, results in attenuated ERK1/2 phosphorylation and reduced HGF-dependent biological responses that depend on ERK activation, such as cell migration [30]. Similarly, following activation of Met by HGF, the small GTPase Rac1 becomes activated by the Rac-specific GEF Tiam1 on Rab5 vesicles [26]. The recycling of activated Rac1 back up to the plasma membrane, possibly through Rab4-dependent vesicles, is required to direct actin-remodeling during cell migration [26]. In this way, RTK-mediated recycling can sustain activation of signaling complexes formed both on endosomes and at the plasma membrane. As recycling leads to increased receptor stability, a further consequence of recycling is that more receptors would be predicted to be available for ligand engagement and further rounds of signaling. In addition to RTKs, recycling of the beta2 adrenergic receptor (B2AR) through a Rab4 pathway is also selectively controlled in a sequence-dependent manner distinct from bulk recycling. Here, a PDZ-interacting domain within the C-terminus of the B2AR directs its recycling through a subset of actin-stabilized tubular extensions [32,33]. Positive selection of B2ARs into recycling tubules requires SNX27 and the retromer complex [32]. Importantly, RNAi targeted loss of the retromer sorting protein VPS35 mistargets the B2AR towards the degradative pathway in a similar manner to GGA3 knockdown on Met. This results in a reduction in ligand, isoproterenol-mediated downstream cAMP signaling [32]. Additionally, recycling of MHC class I requires recruitment of the common tubule component EHD1 to early endosomes by the small GTPase Rab35, which becomes activated by the Rab35 GEF, connecdenn [34]. Although previous reports had implicated Rab35 in transferrin receptor recycling [35], neither transferrin nor Beta1 integrin recycling, which have also been shown to utilize a similar recycling pathway to MHC class I, were affected by Rab35 [34], suggesting that recruitment of Rab35 may mediate cargo-specific recycling. To date, positive selection for other cargo into recycling pathways, following ligand activation, remains unclear. The ligand dose and internalization route can influence whether the EGFR is degraded or recycled, with low EGF associated with increased recycling and signaling compared to high EGF [9]. However non-ubiquitinated EGF–EGFR complexes can recycle through a kinetically short or long recycling pathway, similar to the transferrin receptor [6]. In contrast to EGF, which remains bound to EGFR in endosomes, transforming growth factor alpha 233

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(TGF-a) becomes uncoupled from EGFR in the acidic environment of the early endosome. This results in decreased targeting of EGFR towards the degradative pathway, promoting enhanced EGFR recycling [36,37]. TGF-a is a more potent mitogenic ligand than EGF, and high TGF-a expression correlates with highly aggressive breast tumors [38]. Hence for certain receptors such as the Met and B2AR receptors, trafficking decisions occur at the level of the early endosome, and depend on specific ligand-dependent recruitment of adaptor proteins, suggesting that different receptors may be ‘hardwired’ to traffic through distinct pathways. In this regard, trafficking may be viewed as an inherent method for differentially regulating multiple receptors that activate similar signaling pathways. Spatial restriction of receptors Recycling acts to concentrate RTKs and their associated signaling complexes to specific spatially localized subcellular domains (Figure 2). For example, in Drosophila melanogaster, several endocytic proteins including the Cbl ubiquitin ligase and Rab11 are required for the polarized distribution of active RTKs during border cell migration [39,40]. Following HGF-mediated Met activation, Met is internalized and rapidly recycled in a Rab4-dependent manner to apical dorsal ruffles in polarized epithelial cells,

and to lamellipodia in non-polarized cells, where Met engages with downstream signaling complexes [30,41]. Interestingly, in the former case, basolateral recycling to the apical surface results in bulk internalization of Met through dorsal ruffles and subsequent degradation [41]. By contrast, in non-polarized cells, Met recycling to lamellipodia stabilizes this pool of the Met receptor, suggesting cell type-specific requirements for Met signal duration. Met recycling and targeting to lamellipodia requires GGA3 and the small GTPase Arf6, and the ablation of GGA3 or Arf6 abrogates lamellipodia formation in response to HGF and reduces cell migration and invasion [30,42]. Arf6 is also required for recycling of the small GTPase Rac to dorsal ruffles in response to HGF [26] and localizes to and is required for invadopodia formed by invasive melanoma and breast cancer cells [42,43]. Lamellipodia, dorsal ruffles and invadopodia are all dynamic actin-dependent structures involved in cell migration and invasion. Arf6, through its ability to modulate trafficking in response to HGF, is an important regulator of epithelial cytoskeleton remodeling, and these studies highlight the requirement for spatially restricted signaling for cell migration and invasion downstream from HGF. In most epithelial tissues, E-cadherin molecules form cell–cell adhesions at lateral surfaces. These adhesions

(b)

(a)

(c)

E-cadherin β−catenin α−catenin

TGN

Ub

p120

AP-2

Arf6

Dorsal ruffle

Rab8

Rab5 Tiam1

P

Rac1 GDP

Cortactin

GTP

MT1-MMP Exocyst

IQGAP1

Hakai

(d)

VAMP7 Rab4 Met α5β1

Adherens junction

Rab25

GGA3 RCP EGFR

Lamellipodia

Invadopodia

TRENDS in Cell Biology

Figure 2. Examples of spatially restricted trafficking. (a) Epithelial cell–cell contacts contain the adherens junction protein complex of E-cadherin, p120 catenin (p120), acatenin and b-catenin. Upon displacement of p120, AP-2 and Hakai are recruited to promote E-cadherin endocytosis. (b) During growth factor stimulation, some cells form apical protrusions called dorsal ruffles. Endocytic recycling of Rac1 to these dorsal ruffles requires the Rac1 GEF Tiam1 and Arf6 on Rab5 endosomes in response to hepatocyte growth factor (HGF). (c) MT1-MMP trafficking to sites of invadopodia involves exocytosis though the SNARE, VAMP7, as well as the exocyst complex. Cortactin and IQGAP1 also regulate MT1-MMP trafficking. (d) Lamellipodia. The Met receptor recycles through a Rab4/GGA3 pathway to the leading edge of cells during HGFmediated cell migration. The integrin a5b1 and EGFR are targeted to the leading edge of cells through a Rab25/RCP complex.

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Table 1. Protein aberrations found in cancer associated with defective trafficking Protein EGFRvIII

Aberration Deletion of aa 6-273 in extracellular domain

Cancer Glioblastoma, non-small cell lung (NSCLC), breast, prostate, ovarian, head and neck Lung and gastric

Met Met

Splice site deletion of exon 14 M1268T and D1246N

E-cadherin

V832M

Cbl

R420Q

AML

EGFR

L858R, L858R/T790M D746-50

NSCLC

TGF-b receptor II p53

E221V/N238I R175H and R273H

Oral squamous cell carcinoma (OSCC) Several cancers

HIP1

Overexpression

Prostate and breast

Cortactin

Overexpression

HAX1

Overexpression

Head and neck squamous cell carcinoma (HNSCC) Advanced OSCC

Rab25

Overexpression

Ovarian and breast

Rab25 RCP

Underexpression Overexpression

Colon cancer Breast cancer

HER2/ErbB2

Overexpression

CLIC3

Overexpression

Rabaptin-5

Underexpression

NDRG1

Underexpression

Breast, ovarian, stomach, bladder, salivary and lung cancer Ovarian and pancreatic cancer Primary clear-cell renal carcinoma (CCRCC) and breast Prostate and breast cancer

Papillary renal carcinoma Hereditary gastric cancer

Trafficking defect Inefficient internalization, ubiquitination and degradation; increased endocytosis and trafficking of heterodimer partners wtEGFR and ErbB2 Impaired Cbl recruitment and Met RTK degradation Increased endocytosis/recycling of the Met RTK Impaired basolateral transport of E-cadherin, intracellular accumulation Impaired FLT3 RTK ubiquitination and endocytosis Impaired endocytosis, Cbl-mediated ubiquitination, increased HER2 heterodimerization Impaired TGF-bRII endocytosis, E-cadherin localization Increased a5b1 integrin and EGFR recycling

Experimental system Gliomaa, breasta, ovariana cell lines

Ref. [86–90]

NSCLC cell lines

[61,62]

NIH 3T3a cell line

[64]

Dog and pig kidneya epithelial cell lines

[46]

Murine myeloid

a

cell line

[91]

Lung cancer cell line

[92]

OSCCa cell line

[93]

Lung cancera, retinal epithelial (RPE)a and mammary epitheliala cell lines NIH 3T3a cell line

[78]

HNSCC cell lines

[95]

OSCC cell lines

[96] [71,73,75]

Altered b1 integrin trafficking Increased a5b1 integrin and EGFR recycling Increased EGFR recycling, decreased degradation and/or impaired endocytosis

Ovarian carcinomaa, immortalized mammary epitheliala, breast cancera cell lines Rab25-deficient mice mammary epitheliala, breast cancera, ovarian cancer cell lines Mammary epitheliala, murine myeloida, NIH 3T3a, breast cancer cell lines

Increased recycling of a5b1

Ovarian cancera cell lines

[76]

Impaired EGFR degradation

CCRCCa, osteosarcoma, breast cancer and cervical cancer cell lines Prostate cancer cell lines

[68]

Altered AP-2 levels, redistribution of clathrin and upregulation of EGFR, transferrin receptor, FGFR-3 and FGFR-4 Impaired Cbl recruitment and EGFR downregulation Impaired avb6 integrin endocytosis Increased a5b1 integrin and EGFR recycling

Impaired E-cadherin recycling

[94]

[97] [73,77,78] [98–100]

[101]

a

Exogenous protein expression.

maintain epithelial cell polarity, act as mechanosensors and signal for contact growth inhibition [44,45]. The importance of E-cadherin in cancer is underscored by its frequent mislocalization or loss in epithelial tumors [44]. For example, a germline mutation in E-cadherin found in hereditary gastric cancer occurs in a region containing a PIPKIg binding site, leading to impaired AP-1B-mediated basolateral transport of E-cadherin to the plasma membrane (Table 1) [46]. At junctions, E-cadherin localization is dynamic; E-cadherin can be constitutively endocytosed and recycled or, in response to external stimuli, undergo CDE or CIE, leading to recycling or lysosomal degradative

fates [47–49] (for review see [48]). Association of p120 catenin (p120) with the juxtamembrane domain of E-cadherin prevents E-cadherin internalization by physically blocking access to dileucine and tyrosine phosphorylation sites, important for the recruitment of endocytic adaptor AP-2 and the E3 ubiquitin ligase, Hakai, respectively [50,51]. Although the mechanisms involved in sorting Ecadherin from endosomes to lysosomes remains unclear, activation of Met, EGFR or Src leads to recruitment and Hakai-mediated ubiquitination and degradation of E-cadherin [44], and recruitment of SNX1 promotes recycling of E-cadherin [47]. 235

Review Invadopodia formation occurs upon integrin engagement or growth factor stimulation [52] and can be viewed as a specialized microenvironment, requiring polarized trafficking of matrix metalloproteinases (MMPs) in a manner similar to that observed for RTKs and integrins. These enzymes are enriched at sites of invadopodia and the invasive front of tumor cells [52]. Under normal circumstances, MMP levels at the cell surface are low because of their constitutive internalization. However, in tumor cells, invadopodia represent sites where MMP surface levels are elevated because of concomitant decreased endocytosis and increased exocytosis [52]. Association of b1 integrin with MT1-MMP was found to inhibit MT1-MMP internalization in endothelial cells, suggesting that integrins may physically regulate MMP secretion [53]. How MT1-MMP is directed specifically to sites of invadopodia is unclear, but probably involves microtubule-directed targeting and coordination with actin machinery. Cortactin and IQGAP1 have both been localized to invadopodia and may regulate vesicle secretion [54,55]. Additionally, knockdown of components of the exocyst complex (Sec6, Sec8 or Sec10) or SNARE complex (VAMP7) prevents MT1-MMP delivery to invadopodia and supports a role for these complexes in the tethering and targeting of MT1-MMP-containing vesicles to the plasma membrane [55,56]. Dysregulation of trafficking in cancer Altered RTK ubiquitination Upon ligand activation, several RTKs including the EGF and Met receptors recruit the Cbl family of E3 ligases (cCbl, Cbl-b, Cbl-3) and become ubiquitinated [57]. Remarkably, although Grb2-mediated Cbl recruitment is important in RTK endocytosis, receptor ubiquitination per se is not necessary for the internalization of the EGF or Met receptors [6,58,59]. Instead, ubiquitinated receptors are recognized by a series of endocytic proteins containing ubiquitin-interacting domains, including the ESCRT complexes, to actively sort receptors into the intraluminal vesicles of MVBs for their eventual degradation in the lysosome. In the case of the Met receptor, mutation of the Cblbinding site (Y1003F) leads to inefficient recognition of Met by the ESCRT-0 subunit Hrs, decreasing entry of Met into MVBs and increasing Met stability and activation [59]. Prolonged activation of Met results in sustained activation of the Ras–MAPK pathway and is sufficient to promote cell transformation and tumorigenesis [59,60]. Naturally occurring alternatively spliced isoforms of Met harboring deletion of exon 14, comprising the Cbl-binding site, have been identified in lung and gastric cancers [61,62]. These Met mutants, found in primary tumors, show similar enhanced stability of Met in response to ligand and transforming capacity as the engineered Met mutant cell lines, highlighting the importance for Met ubiquitination in vivo to suppress transforming activity. These findings extend to other RTKs, as loss of the Cbl-binding site occurs within several RTK-derived oncoproteins [57]. For example, chromosomal translocations observed in hematological malignancies and solid tumors often harbor constitutively active cytosolic regions of RTKs [57]. These oncogenic chimeric proteins act twofold: they are often cytosolic and are 236

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excluded from the endocytic pathway [63], and often no longer contain their Cbl-binding site, preventing efficient ubiquitination and downregulation [57]. Interestingly, when expressed in fibroblasts that express HGF, weak oncogenic alleles of the Met RTK found in human papillary carcinomas show increased endocytosis and recycling [64]. This is associated with the transforming activity of these mutants in fibroblasts, consistent with prolonged oncogenic Met signaling in Cbl-impaired Met mutant epithelial cells [59]. Up to 25% of breast cancers overexpress HER2/ErbB2, leading to the formation of EGFR/HER2 heterodimers [65]. In contrast to EGFR, other EGFR family members, ErbB2, ErbB3 and ErbB4, do not efficiently engage with Cbl and are only weakly targeted to the lysosome for degradation [66]. Formation of EGFR/ErbB2 heterodimers leads to enhanced EGFR recycling, impaired degradation and sustained signaling [66]. Several therapies aimed at disrupting ErbB2 heterodimerization and/or increasing ErbB2 endocytosis are now being used and continue to be active areas of research. Together, these findings highlight that escape of RTKs from Cbl-mediated downregulation is an important process selected for during tumorigenesis. Altered trafficking by hypoxia Under conditions of low oxygen, tumor cells cope by upregulating RTK-dependent signaling pathways such as those that promote cell survival, proliferation and invasion [67]. Although enhanced RTK signaling can occur through elevated levels of receptor, mediated by transcriptional and translational mechanisms, hypoxia-mediated regulation of RTK trafficking is another mechanism underlying enhanced stability and signaling of EGFR [68]. Under hypoxic conditions, the transcription factor hypoxia-inducible factor (HIF) represses the expression of Rabaptin-5, a Rab5/Rab4 effector [68]. Loss of Rabaptin-5 leads to inefficient early endosomal fusion, resulting in retention of EGFR in the early endocytic pathway and prolonged activation of downstream signaling pathways involving ERK1/ 2 and PI3K/AKT [68] (Figure 3). A role for hypoxia in regulating integrin trafficking has also been reported. In response to hypoxia in tumor cells in culture, microtubules become stabilized, leading to Rab11-dependent transport of the laminin receptor, integrin a6b4, out of a perinuclear compartment leading to its redistribution up to the plasma membrane [69]. Expression of a GDP-bound inactive Rab11-S25N, a dominant negative mutant, decreases invasion of MDA-231 breast cancer cells induced by hypoxia [69], supporting a role for altered trafficking in tumor cell invasion during hypoxic responses. Rab25 in cancer Rab25 is an epithelial-specific member of the Rab11 family of proteins [70]. Rab25 contains a leucine rather than glutamine in the GTP-binding domain, suggesting that it normally exists in a constitutively active GTP-bound state [70]. Amplification of the locus encompassing Rab25 occurs in approximately half of ovarian and breast cancers, as detected by high-density array comparative genomic hybridization (CGH) [71]. Likewise, overexpression of the Rab25 interacting partner, Rab coupling protein

Review

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Hypoxia

(a)

Angiogenesis

(b)

α6β4 integrin

RTK

+VEGF

-VEGF

P

Rab4 Src

P

Rab11 P

Synectin myosin VI

P

Rab5 HIF

PTP1B Rabaptin5

P

P

CD63 Sustained signaling

Degradation TRENDS in Cell Biology

Figure 3. Trafficking mechanisms during hypoxia and angiogenesis. (a) During hypoxia, hypoxia-inducible factor (HIF) represses Rabaptin-5 transcription leading to impaired Rab5-mediated endosome fusion and receptor tyrosine kinase (RTK) downregulation. Increased Rab11-dependent recycling of a6b4 also occurs under hypoxic conditions. (b) In the absence of ligand, the vascular endothelial growth factor receptor VEGFR2 recycles through a Rab4 compartment. Upon ligand binding, VEGFR2 is targeted to a late endosomal compartment and is recycled in a Src-dependent manner. This pathway is supported by synectin and myosin IV and antagonized by PTP1B.

(RCP), frequently occurs in breast cancer and elevation of both Rab25 and RCP is associated with poor clinical outcome [70,72,73]. Ectopic Rab25 overexpression increases tumor development whereas RNAi-mediated decrease of Rab25 levels inhibits tumor growth in vitro and in vivo in ovarian and breast cancer cells [71,74]. In ovarian cancer cells, Rab25 directly interacts with the cytoplasmic tail of the b1 integrin to promote the delivery of the integrin for fibronectin, a5b1, to pseudopodial tips during cell migration and invasion [75]. In certain cases where the chloride intracellular channel protein 3 (CLIC3) is upregulated in Rab25-amplified tumor cells, CLIC3 can promote recycling of activated integrins from late endosomes/lysosomes, thereby diverting them from degradation [76]. This allows conformationally active integrins to be transported to the cell rear while inactive integrins are transported to the cell front during migration [76]. Moreover, RCP coordinates recycling of a5b1, together with EGFR, to membrane protrusions during fibronectin-mediated cell migration [77], supporting cooperative regulation of EGFR and integrin trafficking pathways. Increased RCP-mediated a5b1 integrin and EGFR recycling occurs in tumor cells that harbor inactivating mutations in the tumor suppressor p53, and correlates with enhanced invasion and metastasis [78]. Hence, Rab25 and its associated proteins play an important role in integrin-dependent cancer cell migration and invasion. Altered trafficking in angiogenesis The formation of new blood vessels is essential for growth of solid tumors by increasing the supply of nutrients and oxygen to cancer cells [79]. Proangiogenic factors induce signaling cascades leading to proliferation, migration of epithelial and precursor cells and remodeling of vasculature to form new blood vessels. These factors include the vascular endothelial growth factors (VEGFs) and ligands

containing RGD-binding sites such as vitronectin for endothelial integrins, avb3 and avb5 [79,80]. Interestingly, in the absence of ligand, 40% of the VEGF RTK is constitutively internalized into early, Rab4-positive recycling endosomes, with the rest remaining on the plasma membrane [81] (Figure 3). In response to VEGF stimulation, the VEGF receptor VEGFR2 RTK is retargeted to CD63+ late endosomes, where it is stored or transported in a novel Srcdependent manner to peripheral vesicles that are spatially localized to sites of cell protrusions and cell–cell adhesions [81]. Entry of VEGFR2 into the early endosome requires the PDZ domain scaffold protein synectin and the motor protein myosin VI complex [82]. Disruption of synectin/ myosin VI results in dephosphorylation of VEGFR2 by the PTP1b phosphatase on a key intracellular tyrosine residue (Y1775), resulting in reduced ERK1/2- and PI3K-mediated signaling [82]. Of note, mice and zebrafish lacking myosin IV or synectin exhibit decreased arterial morphogenesis [82], highlighting the significance of VEGFR2 trafficking in vivo. Intriguingly, when used at low doses, the avb3 and avb5 integrin inhibitor cilengitide has been found to cause Rab4-dependent recycling of VEGFR2, enhancing signaling and promoting angiogenesis [83]. As cilengitide has been relatively unsuccessful in clinical trials, these results may help to explain why cilengitide may not be an effective anticancer treatment under certain contexts. Additionally,

Box 1. Outstanding questions  In vivo studies are required to address consequences of altered recycling.  Do integrins signal from endosomes? If so, how does this influence integrin function?  How are signals from RTKs, integrins and angiogenic growth factors integrated during hypoxia and angiogenesis?  Do RTKs traffic with MMPs to invadopodia? 237

Review VEGF-A can activate PKD1 to modulate avb3 integrin trafficking in endothelial cells, suggesting bidirectional crosstalk between VEGF and integrin receptors [84]. Concluding remarks Trafficking is emerging as a prominent aspect of receptor function and biological response. To date, several aspects of receptor trafficking remain unclear, partly because of celltype or experimental discrepancies. In this regard, further in vivo studies are needed to validate the physiological relevance of trafficking mechanisms observed in vitro. Improvements in imaging techniques should also help to more precisely define the spatial and temporal dynamics of receptor trafficking. The demonstration that altered RTK and integrin trafficking promotes tumorigenesis highlights the need to better understand the molecular mechanisms through which this occurs. Coactivation, along with colocalization, of several RTKs and integrins has been observed, but few studies have addressed why or how this occurs (Box 1). The ubiquitin pathway is a promising avenue for the development of cancer drug targets [85]. Elucidating the role of unexplored ubiquitin-modifying enzymes on receptor trafficking and stability is likely to uncover alternative mechanisms derailed by cancer cells. Furthermore, more work is needed to understand how MMP polarized trafficking is achieved. Although RTK signaling influences MMP activity at sites of invadopodia, it is unclear whether RTKs are also present in invadopodia and can directly traffic with MMPs. The demonstration that cancer cells can overcome integrin inhibitors by upregulating the trafficking pathways of other signaling receptors [83] underscores the need to better understand mechanisms by which cancer cells dynamically modulate the trafficking network. Acknowledgments We thank the anonymous reviewers for helpful suggestions. We apologize for any omissions when citing relevant literature due to space restrictions.

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