Articles in PresS. Am J Physiol Endocrinol Metab (February 24, 2009). doi:10.1152/ajpendo.90857.2008
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Breakdown of endocytosis in the oncogenic activation of receptor tyrosine kinases
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Jasmine V. Abella 1,2 and Morag Park 1,2,3,*
Department of Biochemistry1, Medicine3, and Oncology4, McGill University, Montréal, Québec, Canada; Molecular Oncology Group2, McGill University, Montréal, Québec H3A 1A1, Canada 2. Running title: RTKs and cancer: escape from endocytic regulation *Corresponding Author. Mailing address: Rosalind and Morris Goodman Cancer Centre, Rm 511, 1160 Pine Ave West, Montreal, H3A 1A3, Quebec, Canada. Tel: 514-398-5749 Fax: 514-398-6769 E-mail:
[email protected].
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1 Copyright © 2009 by the American Physiological Society.
1
Abstract
2
There is increasing evidence to support the concept that the malignant behavior of many tumors is
3
sustained by the deregulated activation of growth factor receptors. Activation of receptor tyrosine kinases
4
(RTKs) by their respective ligand(s) initiates cellular signals which tightly modulate cell proliferation, survival,
5
differentiation and migration to ensure normal tissue patterning. Uncontrolled activation of such signals can
6
therefore have deleterious effects leading to oncogenesis. To date, deregulation of most RTKs have been
7
implicated in the development of cancer although the mechanisms which lead to their deregulation are not yet
8
fully understood (10). RTK endocytosis, the internalization and trafficking of receptors inside the cell, has long
9
been established as a mechanism to attenuate RTK signaling. However, RTKs have been demonstrated that to
10
continue to signal along the endocytic pathway, which contributes to the spatio-temporal regulation of signal
11
transduction. This review will focus on recent advances linking defective endocytosis of RTKs in the
12
development of cancer.
13
2
1
Introduction
2
RTKs are single pass transmembrane proteins whose intrinsic catalytic activity, to phosphorylate tyrosine
3
residues, is regulated through ligand binding to the extracellular domain of the protein. Engagement with ligand
4
promotes receptor dimerization or oligomerization, inducing conformational changes within the catalytic
5
domain that allow binding of ATP and activation of the enzyme. Kinase activation first promotes
6
phosphorylation of tyrosine residues within the activation loop of the catalytic domain, which helps to stabilize
7
the activation state (66). The subsequent transphosphorylation of specific tyrosine residues located outside of
8
the catalytic domain, generates phosphotyrosine dependent docking sites for proteins that contain SH2 or PTB
9
domains. These domains recognize phosphotyrosine residues in the context of their surrounding amino acids
10
(125) promoting the formation of a ligand dependent signaling complex. Proteins recruited to RTKs, include
11
those with enzymatic activity, such as phospholipase C gamma (PLCγ), phosphatidylinositol 3' kinase (PI3K)
12
and cytoplasmic tyrosine kinases of the Src superfamily. In addition, adaptor and scaffold proteins that lack
13
enzymatic activity, act to assemble and recruit networks of signaling proteins to the RTK through the presence
14
of additional protein-protein interaction domains (124). These signaling complexes relay and amplify signals
15
that ultimately lead to changes in gene transcription, as well as non-transcriptional changes that promote
16
remodeling of the actin cytoskeleton to alter cell shape, motility and adhesion (124).
17
Multiple mechanisms lead to the oncogenic activation of RTKs. These include receptor amplification or
18
transcriptional over-expression, chromosomal translocation, point mutation and the formation of an autocrine
19
loop (85). Of the 58 genes encoding RTKs (10), somatic mutations have now been identified in each of these in
20
human cancer (47). Many other studies have also begun to catalogue the expression of RTKs, correlating
21
changes in expression with human malignancies (111, 173). However, it still remains to be tested whether these
22
mutations or alterations in expression are selected for and contribute to tumor formation, or are simply
23
passenger mutations. Genomic amplification, over-expression or mechanisms that inhibit receptor degradation,
24
can result in an increased concentration of RTK levels at the cell surface. This in turn can lead to enhanced
25
dimerization and activation of the receptor in the absence of ligand, as well as increased sensitivity to low levels
26
of ligand. Members of the ErbB/epidermal growth factor receptor (EGFR) family are commonly over-expressed
27
in human tumors where ErbB2/HER2 is amplified in up to 20% of human breast cancers (114) and the
28
ErbB1/EGFR in 80% of head and neck tumors, 40-50 % of giloblastomas, and less than 20% of various
29
squamous cell carcinomas (46, 187, 192). Chromosomal translocations, which are prevalent in hematological
30
malignancies, but also occur in solid tumors, generally fuse a protein dimerization motif to the cytosolic kinase
31
domain of a RTK (106, 141, 161). This removes the extracellular ligand binding domain of the RTK, but results
3
1
in a constitutively active receptor, mediated through protein dimerization in the absence of ligand (141).
2
Chromosomal translocation of the platelet derived growth factor receptor beta (PDGFRβ) with the dimerization
3
motif of Tel, a member of the ETS family of transcription factors, results in constitutive activity of the receptor
4
and is associated with chronic myelomonocytic leukemia or chronic myelogenous leukemia (44, 106). Tumor
5
cells are also found to express both the RTK and its cognate ligand, resulting in continuous receptor activation.
6
In human breast carcinomas, the Met RTK and its ligand, the hepatocyte growth factor (HGF) are both
7
expressed in the tumor cells and this autocrine loop correlates with the histological grade of the tumor and
8
reduced survival (28). The co-expression of EGFR with one of its ligands, EGF or transforming growth factor
9
alpha (TGFα), in primary lung adenocarcinomas correlates with reduced survival rates of patients (168).
10
Similarly, high co-expression of EGFR family ligands (TGFα, EGF and amphiregulin) correlates with larger
11
tumor diameter and high histological grade in a large number of breast cancers, highlighting the importance of
12
this RTK family in tumor progression (137). Point mutations occur in both the cytosolic domain as well as the
13
extracellular domain of RTKs. These can activate RTKs through a variety of mechanisms. These include
14
mutations that promote receptor dimerization in the absence of ligand (65, 95), mutations that relieve auto-
15
inhibition of the kinase domain allowing catalytic activity in the absence of ligand (67) or increasing sensitivity
16
to ligand (65). Recently, it has become clear that mutations that uncouple RTKs from down-modulation,
17
resulting in delayed ligand induced degradation of RTKs, are associated with numerous cancers, underscoring
18
the importance of this mechanism of regulation of RTKs (129).
19 20
TERMINATION OF RTK SIGNALING
21
Activation of a RTK on the plasma membrane by ligand initiates both a signal transduction cascade as
22
well as mechanisms to modulate receptor signaling and down-regulate the RTK. These involve receptor
23
dephosphorylation as well as entry into the endocytic trafficking pathway, in general for subsequent degradation
24
in the lysosome (1, 183).
25 26
Regulation of RTKs through Protein Tyrosine Phosphatases. Protein tyrosine phosphatases (PTPs) can
27
regulate RTK signals in both a positive and negative manner. The PTP SHP-2 is essential for growth factor
28
induced activation of the MAPK pathway (104, 116), whereas dephosphorylation of the insulin receptor or Met
29
RTK by PTP-1B negatively regulates receptor signaling (34, 79, 150). PTPs also regulate RTKs in a ligand-
30
independent manner, by keeping receptors inactive at the plasma membrane (74, 138) and PTP-1B has been
31
shown to dephosphorylate the Flt3 RTK (Fms-like tyrosine kinase 3) as it traffics through the synthetic pathway
4
1
(155). Although it is clear that PTPs are important physiological regulators of RTKs, they may play a more
2
important role in modulating signal intensity than in terminating signals. Interestingly, internalization of the
3
EGFR from the plasma membrane is required for dephosphorylation by PTP-1B, which is tethered to the
4
endoplasmic reticulum (50), demonstrating that EGFR internalization and dephosphorylation are coordinated
5
processes. The Met RTK is dephosphorylated prior to being degraded, suggesting that dephosphorylation is a
6
regulated event and not merely the consequence of receptor degradation (150). However, whether PTPs directly
7
regulate endocytosis of RTKs still remains to be determined.
8 9
Down-regulation of RTKs through the endocytic pathway. The endocytic pathway was established to act as the
10
major mechanism for down-regulation of the EGFR (182). Although several distinct pathways for RTK
11
internalization have been identified (107), these are as yet not extensively studied for RTKs and for the
12
purposes of this review, we shall focus solely on the clathrin-dependent pathway. Following ligand stimulation,
13
internalized receptors are subject to two distinct fates: 1) recycle back to the plasma membrane or 2)
14
degradation via the lysosomal pathway (Figure 1). The first identified and best-studied route for entry of RTKs
15
into the cell is the clathrin-dependent pathway. Briefly, receptor-ligand complexes are recruited by adaptor
16
molecules such as AP-2, Eps15 and CIN85 into clathrin-coated pits (CCPs) within the plasma membrane
17
(Figure 1). These pits eventually bud into the cell to form clathrin-coated vesicles (CCVs) through a scission
18
event catalyzed by the enzyme dynamin. CCVs shed their clathrin coat and deliver receptors to the endosomal
19
network. At this point, receptors may become uncoupled from their ligand due to a decrease in pH and recycle
20
back to the plasma membrane or progress down the endocytic pathway to be sorted for lysosomal degradation
21
(42). The rapid removal of RTKs from the cell surface and the subsequent targeting to lysosomal compartments
22
is important to prevent sustained activation from both the plasma membrane (182) and on endocytic vesicles
23
(23), which can lead to cell transformation (103) and tumorigenesis (129).
24 25
Ubiquitin. Historically, protein ubiquitination has been demonstrated to target proteins for degradation by the
26
26S proteasome (37). It is now clear however, that ubiquitin plays a pivotal role in the endocytic pathway to
27
target RTKs for lysosomal degradation. Protein ubiquitination can have several functions based on the type of
28
ubiquitin chain attached (185). Ubiquitin chains are formed through the subsequent addition of ubiquitin
29
moieties onto specific lysine residues on ubiquitin. Ubiquitin chains formed through the addition to lysine 63,
30
(K63), adopt an extended linear conformation that has been demonstrated to have non-proteolytic functions and
31
act as a protein scaffold (27, 176). Lysine 48 (K48) linked chains have a very different topology, adopting a
5
1
closed conformation (29). Whereas K48 linked ubiquitin chains are a signal for proteasomal degradation, K63
2
chains and mono or multi-mono-ubiquitination of proteins is not. Mono-ubiquitin and K63-linked chains act as
3
a docking sites for proteins which contain ubiquitin interacting domains or motifs (185). The discovery of
4
domains able to bind to ubiquitin is expanding rapidly, bringing the current total to 16 (69). It is evident that
5
different domains have different affinities for the type of ubiquitin linkage and this undoubtedly provides an
6
important level of regulation in the ubiquitin-signaling pathway, which parallels that of protein phosphorylation
7
(185).
8
Initial studies in yeast demonstrated that receptor ubiquitination is required for internalization of
9
transmembrane proteins (58, 81, 142). Subsequently, using the cell surface uracil permease transporter as a
10
model, it was established that although mono-ubiquitination was sufficient to induce transporter endocytosis,
11
K63-linked polyubiquitin chains were required for efficient endocytosis and transporter turnover (43). Later
12
studies in mammalian cells however, proved that receptor ubiquitination is not essential for internalization (2,
13
26, 62) but may regulate this process (158). Instead, in mammalian cells, mono-ubiquitination or K63-
14
polyubiquitination of RTKs is required to target them for efficient lysosomal degradation (7, 12, 49, 63, 105,
15
110, 177). Both mono-ubiquitin and K63-linked ubiquitin chains, act as sorting signals that are recognized by
16
proteins of the ESCRT (endosomal sorting complex required for transport) complex, which contain ubiquitin
17
interacting motifs (UIM) (184, 185). ESCRTs recruit and orchestrate receptors for internalization into the
18
endosomal lumen, creating a multi-vesicular body (MVB) (Figure 1) (17). For example, Hrs (Hepatocyte
19
Growth Factor regulated tyrosine kinase substrate) and STAM (Signal Transducing Adaptor Molecule) are part
20
of the ESCRT-0 complex and both contain UIMs (59, 174). These recognize ubiquitinated RTKs and recruit
21
them onto a specific domain on the endosomal membrane, defined by a flat bilayered clathrin lattice (147)
22
(Figure 1). Once recruited onto this limiting membrane, ubiquitinated receptors are processed by ESCRT
23
complexes I to III, which contain several ubiquitin binding proteins including, TSG101 (Tumor Susceptibility
24
Gene 101) as well as others (167). ESCRT complexes can recruit deubiquitinating enzymes, which may be
25
required to remove ubiquitin from RTKs prior to their internalization into the endosomal lumen (4, 97, 144).
26
Upon internalization into the MVB, fusion with lysosomes results in RTK degradation through the action of
27
lumenal acid-dependent proteases (76, 132). Disruption of RTK ubiquitination or of the ESCRT complex,
28
therefore, has the potential to enhance receptor stability, prolong signaling and induce cell transformation.
29 30
Role of Cbl in RTK down-regulation. The E3-ubiquitin ligase Cbl, acts as a key negative regulator of RTKs,
31
promoting the ubiquitination of activated receptors to target them for efficient internalization into MVBs and
6
1
subsequent lysosomal degradation. In mammals, the Cbl family of ubiquitin ligases consists of c-Cbl, Cbl-b
2
and Cbl-3 (115). All three members are highly homologous in their amino-terminus, which consists of a
3
tyrosine kinase binding (TKB) domain, containing a variant SH2 domain which engages with specific
4
phosphotyrosine residues in RTKs. A RING domain follows the TKB and mediates transfer of ubiquitin to the
5
RTK (169). Recruitment of Cbl to RTKs leads to Cbl phosphorylation which is required for efficient E3-ligase
6
activity (93).
7 8
Loss of function of Cbl proteins in human cancer. Cbl was first identified as a retroviral oncogene (v-Cbl)
9
from the Cas NS-1 retrovirus, which induces pre-B-cell lymphomas and myelogenous leukemia in mice (86).
10
Mutations in Cbl in general, act to disable the ubiqitin ligase activity, yet allow the recruitment of Cbl to its
11
substrates such as RTKs. Such mutations uncouple RTKs from Cbl mediated ubiquitination and efficient
12
lysosomal degradation. Several distinct mutations in Cbl have now been identified in patients with acute
13
myeloid leukemia, acute non-lymphocytic leukemia (ANLL) and in an ANLL transgenic mouse model (11, 152,
14
159). These mutations all cluster around exon 8, resulting in missense mutations in the linker-region or deletion
15
of a portion of the RING finger and result in impaired ubiquitin ligase activity (152). Where studied, expression
16
of these mutant Cbl proteins, induce a ligand-independent activation of the Flt3 RTK by inhibiting its
17
ubiquitination and internalization (152).
18
In addition to mutations within Cbl, several mechanisms that sequester Cbl from RTKs have also been
19
identified in cancer (Figure 1). The small GTPase Cdc42, involved in actin remodeling, can also inhibit Cbl
20
mediated EGFR down-regulation through sequestration of Cbl via a complex involving Beta-Pix (190). The
21
adaptor protein Sprouty 2, is phosphorylated upon EGF stimulation and binds to the c-Cbl TKB domain (38, 51).
22
This interaction sequesters Cbl from activated EGFRs, decreasing RTK ubiquitination and internalization and
23
promoting sustained EGF induced MAPK activation (30, 38, 48, 146). Cortactin, which links the actin
24
cytoskeleton to the endocytic machinery, is over-expressed in breast and head and neck carcinomas due to the
25
frequent amplification of the chromosome 11q13 region in these malignancies (123, 156). Over-expression of
26
cortactin in HeLa cells result in a marked inhibition of Cbl mediated ubiquitination and down-regulation of the
27
EGFR, correlating with a reduction in Cbl phosphorylation and recruitment to the EGFR (172). Moreover,
28
depletion of cortactin in head and neck squamous cell carcinoma cell lines accelerated EGFR down-regulation
29
and attenuated MAPK signaling (172). Members of the EGFR family are frequently activated in cervical
30
cancers induced by human papillomavirus type (HPV) (77). Interestingly, E5, an oncoprotein encoded by
31
HPV16, can form a complex with the EGFR (70) and has been demonstrated to enhance the activation and
7
1
signaling of the EGFR through several mechanisms including disruption of the endocytic pathway (165, 171)
2
and more recently, by preventing Cbl recruitment and ubiquitination of the EGFR, resulting in delayed down-
3
regulation of the receptor (197). Finally, several other proteins have also been shown to sequester Cbl away
4
from mediating RTK down-regulation by inducing Cbl degradation. The CD28 receptor enhances T-cell
5
receptor signaling by enhancing the down-regulation of both c-Cbl and Cbl-b (3, 198). The tyrosine kinases Src
6
and Hck, can also interact with c-Cbl resulting in the ubiquitination and degradation of both proteins (6, 61,
7
196). Cbl family members can also interact with the HECT E3 ligases Nedd4 and AIP4 (Itch), which result in
8
proteasomal degradation of Cbl (18, 99).
9 10
RTK mutations in human cancer uncouple Cbl mediated ubiquitination
11
Several RTKs, including the epidermal growth factor (EGF), hepatocyte growth factor/Met, platelet
12
derived growth factor (PDGF), colony stimulating factor-1 (CSF-1) and stem cell factor/ c-Kit receptors recruit
13
the E3-ligase Cbl upon activation and are subsequently ubiquitinated (35, 75, 88, 94, 108, 179, 186, 195). All of
14
these RTKs have been implicated in the development of cancer and in several cases, oncogenic activation of
15
these RTKs in human cancers can be linked to loss of receptor ubiquitination. The TKB domain of Cbl is
16
recruited to a phosphotyrosine residue of the Met, EGF and c-Kit receptors (93, 105, 128). Substitution of this
17
tyrosine for a phenylalanine residue uncouples direct Cbl binding to these receptors and inhibits receptor
18
ubiquitination (105, 128, 181).
19 20
Met/HGF RTK in lung cancer. In the case of the Met receptor, loss of Cbl mediated ubiquitination results in a
21
receptor that can still internalize but is not efficiently degraded and is now transforming (2, 128). This non-
22
ubiquitinated mutant receptor (Y1003F) is no longer targeted to the ESCRT complex and instead remains on the
23
endosomal membrane where it leads to sustained activation of the Ras-MAPK pathway (2). Importantly, several
24
somatic intronic mutations have been identified in non-small-cell lung cancers that result in the excision of exon
25
14 encoding the Cbl-TKB binding site and are subsequently poorly ubiquitinated (82, 98, 122)}.
26 27
EGFR family in glioblastoma. In a similar manner to the Met RTK, uncoupling the EGFR from Cbl mediated
28
ubiquitination by loss of the Cbl binding site (Y1045F), induces stronger mitogenic signals when compared to
29
the ubiquitinated wild-type receptor (181). The EGFR receptor is amplified in up to 40% of glioblastomas, and
30
in many cases the amplified EGFR bears mutations (31, 33, 188). One common alteration involves truncation of
31
the EGFR (EGFRvV) generating a receptor that lacks the Cbl-TKB binding site at Y1045 (41). The most
8
1
common mutation, deletion of exons 2-7, located in the extracellular domain, generates a truncated EGFR
2
(EGFRvIII), which no longer binds ligand and lacks the dimerization arm of the EGFR (84, 166). However,
3
many studies have demonstrated that this receptor is constitutively phosphorylated, albeit to weaker degree
4
when compared to ligand stimulated wild type EGFR (32, 64, 154) and can induce cell transformation in vitro
5
and in vivo (8, 113, 117). Several reports have shown that the EGFRvIII is uncoupled from Cbl-mediated
6
ubiquitination and/or exhibits low rates of internalization (45, 52, 64, 154). The direct Cbl binding site Y1045
7
was shown to be hypo-phosphorylated thereby compromising Cbl recruitment and receptor ubiquitination (52).
8
Although one study supports that EGFRvIII is appropriately down-regulated by the Cbl family of E3-ligases
9
suggesting that an alternative mechanism of oncogenic activation may be responsible (20). In addition,
10
EGFRvIII was unable to induce phosphorylation of adaptor molecules important for EGFR internalization (e.g.
11
Eps15), resulting in enhanced retention on the plasma membrane, and delayed degradation when compared to
12
the wild-type EGFR (45). The low but constitutive autophopshorylation-dependent signaling of the EGFRvIII
13
coupled with inefficient down-regulation could explain the transforming ability of this variant receptor. EGFR
14
mutants bearing various deletions in the C-terminal tail, where Cbl is recruited, have also been identified in
15
glioblastomas (41) and these receptors would therefore be predicted to be poorly ubiquitinated and inefficiently
16
down-regulated.
17 18
EGFR family in breast cancer. Under physiological conditions, Cbl is poorly recruited to other EGFR family
19
members (HER/ErbB2, 3, 4), which are not targeted for efficient degradation in the lysosome (9, 92, 131).
20
ErbB2, for which there is no known ligand, forms heterodimers with other EGFR family members, the preferred
21
partner being the EGFR. ErbB2 is considered endocytosis impaired, however the mechanism behind this defect
22
is still under debate. Several studies demonstrate that ErbB2 is retained at the plasma membrane (9, 60, 91, 162),
23
whereas others have reported rapid recycling of ErbB2 (5, 16, 55, 56, 80, 89). Since ErbB2 is amplified in
24
human cancers such as breast and ovary (36, 114), the ability to stimulate down-regulation of ErbB2 as a
25
therapeutic strategy has been an active area of research. The discovery that treatment of tumor cells over-
26
expressing HER2 with anti-HER2 antibodies lead to a decrease in cell growth (25) ignited research into this
27
field. Many studies have now shown that monoclonal antibodies can enhance receptor down-regulation through
28
the endocytic pathway (21, 153) possibly by promoting an interaction between Cbl and HER2 resulting in
29
receptor ubiquitination and degradation (80). Herceptin (Trastuzumab), is an example of a humanized
30
monoclonal antibody against HER2, which is currently being administered with chemotherapy in the treatment
31
of metastatic breast cancer patients (68). The chaperone Hsp90 interacts with ErbB2 and this association retains
9
1
the receptor at the plasma membrane (15). Pharmacological inhibitors of Hsp90 such as Geldanamycin, have
2
been effective in increasing ErbB2 down-regulation by inducing recruitment of the E3-ligase CHIP to ErbB2
3
and inducing its ubiquitination and cleavage (90, 91, 191, 199). Geldanamycin is currently being evaluated in
4
clinical trials. Importantly, the combined use of Trastuzumab with an Hsp90 inhibitor induced recruitment of
5
both Cbl and CHIP E3-ligases resulting in higher levels of ErbB2 ubiquitination and degradation than with
6
individual treatments (133).
7
Amplification of ErbB2 drives the formation of EGFR/ErbB2 heterodimers. This reduces ligand induced
8
endocytosis and degradation of the EGFR and leads to enhanced EGFR levels at the plasma membrane (53, 112,
9
180, 189). Mechanistically, EGFR/ErbB2 heterodimers lead to a reduction in Cbl recruitment, enhance receptor
10
recycling thereby decreasing the efficiency of lysosomal degradation of the EGFR (89, 112). Ligand activated
11
EGFR/ErbB2 heterodimers also show decreased capacity to induce the formation of clathrin-coated pits,
12
resulting in decreased rates of internalization and prolonged EGFR signaling at the plasma membrane (53). The
13
retention of the complex at the plasma membrane may also prevent receptor dephosphorylation by tyrosine
14
phosphatases present on endo-membranes, thereby promoting sustained EGFR activation (120). In contrast,
15
EGFR homodimers, which recruit and become actively ubiquitinated by Cbl, have a greater propensity to
16
continue down the endocytic pathway and are targeted for degradation in the lysosome (89, 189).
17
ErbB2/ErbB3 partnership in breast cancer may be important for the aggressive nature of cancers with ErbB2
18
amplification, due to the increased retention of the ErbB2/ErbB3 complex at the plasma membrane by ErbB2
19
coupled with the enhanced mitogenic signaling of ErbB3. Many therapeutic strategies are now based on
20
disrupting heterodimerization of the endocytosis deficient receptor ErbB2, by using humanized monoclonal
21
antibodies such as pertuzumab (39). These have been effective is rescuing the efficient down-regulation of the
22
EGFR and ErbB3 receptors through the endocytic pathway (39, 148).
The
23 24
Kit and CSF-1 RTKs. In a similar manner to the Met and EGF receptors, deletion of a Cbl TKB binding site
25
(Y568) greatly enhances the transforming and mitogenic activity of the stem cell RTK (c-Kit) (57). Many
26
mutations in the juxtamembrane domain of c-Kit identified in human gastrointestinal stromal tumors (GISTs),
27
relieve auto-inhibition by the juxtamembrane domain resulting in ligand-independent activation of the receptor
28
(14). Such mutations would also result in loss of Cbl recruitment and impaired down-regulation, which may
29
contribute to the oncogenicity of these mutant receptors. Loss of the Cbl TKB binding site has also been
30
implicated in the oncogenic deregulation of the colony-stimulating factor-1 receptor (CSF-1R) in
31
myelodysplasia and acute myeloid leukemia (139) where mutation of the Cbl TKB binding site (Y698),
10
1
enhances the transforming activity of the CSF-1R (143). Notably, the EGFR, c-Kit and CSF-1R were first
2
identified as the transforming agent of oncogenic retroviruses v-erb-B, v-Kit and v-Fms respectively (24, 54,
3
193). These viral oncogenes lack the tyrosine residues required for recruitment of Cbl (102, 129, 186) indicating
4
that mutations found in RTKs which uncouple them from Cbl mediated down-regulation, is an evolutionary
5
conserved pathway that is selected for during tumorigenesis.
6 7
Chromosomal Translocations; mistargeting of RTKs from the endocytic pathway.
8
Chromosomal translocations resulting in ligand independent activation of RTKs occur most frequently
9
in hematologic malignancies (106) but also in solid tumors (161). The PDGFR, FGFR1, Trk and RET receptor
10
families are frequent targets of chromosomal translocations (106). In general, these events fuse the catalytic
11
domain of RTKs with a dimerization motif derived from another gene. This promotes constitutive dimerization
12
and activation of the kinase in the absence of ligand (141). With the exception of FIG-ROS, which is targeted to
13
the Golgi (13), all RTK-derived fusion proteins, where studied, have lost their amino-terminal signal peptide
14
and transmembrane domain to target them to the plasma membrane. Consequently, these RTK variants are
15
localized to the cytosol. Hence, even if they still recruit Cbl and become ubiquitinated, they fail to enter the
16
endocytic pathway and thus escape down-regulation through lysosomal degradation (Figure 1). For example,
17
one of these chimeric RTK oncoproteins (Tpr-Met), which loses the Cbl TKB binding site following
18
chromosomal translocation, is not reduced in its transforming ability upon the insertion of Cbl-TKB site (101).
19
However, targeting this receptor to the plasma membrane is able to attenuate its oncogenic activity (101), by
20
reconstituting the ability of this chimeric-receptor to enter the endocytic pathway, highlighting the importance
21
of RTK endocytosis in receptor down-regulation.
22 23
Cellular stress and receptor down-regulation
24
Oxidative stress is a common feature of transformed cells and represents an imbalance in redox
25
homeostasis. There is now accumulating evidence that cellular stress signals result in aberrant activation and
26
localization of the EGFR, generating an environment whereby the EGFR no longer undergoes degradation. An
27
increase in free radical concentration can have deleterious effects on cellular functions by damaging DNA
28
(strand breakage), proteins (peptide cleavage) and lipids (lipid peroxidation) (87). Under conditions of high
29
hydrogen peroxide (H2O2) levels, in response to EGF, the EGFR is aberrantly phosphorylated, no longer
30
recruits Cbl and hence becomes uncoupled from ubiquitin-mediated degradation (136). In addition, engagement
31
with Eps15, a component of the internalization machinery that is partly dependent on ubiquitin-mediated
11
1
interactions, is decreased (22). In addition, oxidative stress activates the Src tyrosine kinase, which can promote
2
proteasome-dependent degradation of Cbl (196) and therefore may also contribute to the impaired
3
ubiquitination of the EGFR and endocytic adaptors. Under these conditions, the EGFR remains at the plasma
4
membrane for a prolonged period of time (78) and once internalized, is retained in a peri-nuclear compartment,
5
actively signaling (78). Other cellular stresses, such as UV irradiation or inflammatory cytokines induce serine
6
and threonine phosphorylation of the EGFR on a short segment of its C-terminal tail by the p38 stress-induced
7
kinase (121, 178, 200). Under these conditions, the EGFR is not ubiquitinated nor targeted for degradation, but
8
is internalized. In response to UV irradiation, the EGFR undergoes sustained serine/threonine phosphorylation,
9
is internalized and retained on early endosomes (121, 200). The inflammatory cytokine tumor necrosis factor
10
alpha (TNFα) induces transient serine/threonine phosphorylation of the EGFR, which is rapidly recycled upon
11
receptor internalization (200). Both processes are ubiquitin independent and therefore uncouple the receptor
12
from a degradative pathway. The identification of this alternate mechanism of EGFR induced internalization
13
has important implications for cancer therapy since chemotherapeutic agents which have been shown to activate
14
p38 (96), could induce internalization of the EGFR, making tumors refractory to anti-receptor antibody
15
therapies such as Erbitux and Herceptin that target the extracellular domain of the receptor (200).
16
Activity of the ubiquitin activating and conjugating enzymes, E1 and E2 are also regulated by the redox
17
status of the cell (73, 157). Treatment of tissue with hydrogen peroxide leads to diminished levels of
18
endogenous ubiquitin-protein conjugates (73). This is due to S-thiolation of the active-site sulfydryl groups
19
during oxidative stress, which prevents the formation of E1-ubiquitin and E2-ubiquitin intermediates (119), the
20
first two steps in the ubiquitination pathway. Any decrease in the cellular capacity to ubiquitinate proteins
21
would disrupt multiple cellular processes including RTK down-regulation.
22 23
Disruption of the endocytic machinery
24
In addition to mutations that uncouple RTKs from efficient down-regulation through the endocytic
25
pathway, disruption in any of the components of this pathway could effectively delay the internalization,
26
trafficking and degradation of RTKs. Indeed, there is growing evidence to support a role for aberrant
27
endocytosis in the development of human cancers. For example, the endocytic adaptor, HIP1 (Huntington
28
interacting protein 1) is over-expressed in several human cancers including breast, prostate and colon tumors
29
and is associated with poor clinical outcome (135). HIP1 is a clathrin adaptor, which promotes clathrin
30
assembly during CCP formation (Figure 1). Over-expression of HIP1 in NIH3T3 mouse fibroblasts results in
31
enhanced levels of RTKs at the plasma membrane, most likely by interfering with clathrin mediated
12
1
internalization (71, 134). Under these conditions EGFR levels remained stable and EGF stimulation resulted in
2
sustained activation of both MAPK and PI3K pathways, leading to cell transformation. Another endocytic
3
adaptor protein, Numb, (149, 151) is involved in regulating the endocytosis of several transmembrane proteins,
4
including the Notch receptor, EGFR and beta-integrins (72, 118, 151),(160). Numb is lost in 50% of human
5
primary breast carcinomas due to its proteasomal degradation and results in an increase in Notch signaling (126).
6
Since expression of Numb fragments inhibits EGF and transferrin receptor internalization, it is likely that in
7
tumors where Numb is degraded, EGFR signaling will also be up-regulated (151). Interestingly, several other
8
endocytic proteins, including, Eps15, CALM (clathrin assembly myeloid lymphoid leukemia) and Endophilin II
9
have been identified as chimeric fusion proteins as a result of chromosomal translocations in a variety of human
10
leukemias (19) (Figure 1). Although disruption of endocytosis has not yet been demonstrated to be the
11
mechanism for oncogenic activation, many of these chimeric proteins would no longer be targeted to the correct
12
subcellular location to carry out their normal function. In addition, such chimeric proteins may also act in a
13
dominant negative manner and sequester other important proteins from the endocytic pathway.
14
Under hypoxic conditions in tumors, many RTKs exhibit prolonged activation through an unknown
15
mechanism, contributing to oncogenesis (40, 83, 127). Under conditions of hypoxia, endocytosis of the EGFR
16
becomes attenuated due to inefficient early endosome fusion (194). This occurs through the HIF (hypoxia-
17
inducible-factor) dependent down-regulation in transcription of the rabaptin-5 gene (194), an effector of the
18
early endosome Rab5 GTPase (Figure 1) (164). Importantly, rabaptin-5 RNA levels from human primary clear-
19
cell renal carcinoma and breast tumors with a hypoxic signature were significantly down-regulated compared to
20
normal tissue (194).
21
Several studies in Drosophila have uncovered tumor suppressor roles for components of the ESCRT
22
machinery. Mutant cells for either the protein erupted (TSG101, ESCRT I), vsp25 (ESCRT II component) or
23
dVps4 (ATPase, ESCRT III), lose cell polarity and contain an accumulation of actively signaling ubiquitinated
24
receptors (Notch and Thickveins) on endosomes (109, 140, 170, 175). Aberrant receptor signaling leads to
25
ectopic secretion of cytokine-like molecules that induce proliferation of surrounding wild-type cells.
26
Importantly, when apoptosis of mutant cells is inhibited, these cells begin to over-proliferate, reminiscent of
27
precancerous cells which require a second mutation to develop into a tumor (109, 140, 170, 175).
28 29
Conclusions and Perspectives
30
Deregulation of RTK endocytosis is clearly emerging as a mechanism of oncogenic activation that is selected
31
for in human cancers. Much work has now demonstrated that deviation of RTKs from clathrin-mediated
13
1
endocytosis can lead to cell transformation. However, the number of identified internalization pathways
2
available to RTKs has multiplied over the past decade (107). As yet, we do not have a clear understanding of
3
what signals regulate which pathway is taken and how each pathway impacts on RTK signaling and stability.
4
Furthermore, we know very little of the contribution of each pathway in cancer progression. It is therefore
5
paramount that we develop a better understanding of how RTKs traffic normally and how these processes are
6
disrupted in cancer. This knowledge will benefit us greatly in developing new therapeutic strategies and
7
identifying new potential targets.
8 9 10 11
Acknowledgments
12
We thank members of the Park lab for their comments and critical reading of the manuscript. Morag Park is
13
supported by operating grants from the National Cancer Institute of Canada with money from the Canadian
14
Cancer Society, Canadian Institutes of Health Research and from a Terry Fox New Frontiers Group grant. She
15
holds the Diane and Sal Guerrera Chair in Cancer Genetics. Jasmine Abella was supported by a fellowship from
16
the US Department of Defense Breast Cancer Research Initiative (DAMD17-99-1-9284).
17 18
14
1
Figure Legend
2
Figure 1. RTK down-regulation through the endocytic pathway. Growth factor activation of RTKs results in
3
receptor phosphorylation and recruitment of the E3 ligase Cbl. Cbl mediates ubiquitination of RTKs and
4
perhaps some endocytic adaptor molecules, which gather RTKs into clathrin-coated pits (CCPs), (Green RTKs).
5
AP-2 (adaptor protein -2), CIN85 (Cbl-interacting protein, 85kDa), Eps15 (EGFR pathway substrate 15).
6
Uncoupling RTKs from Cbl-mediated ubiquitination (Red RTKs) can have moderate to no effect on RTK
7
internalization, depending on the RTK. CCPs bud into the cytosol to form clathrin-coated vesicles (CCVs).
8
CCVs eventually shed their clathrin coat and fuse with early endosomes. RTKs can either be recycled back up
9
to the membrane, particularly if they are not ubiquitinated, or continue down the endocytic pathway.
10
Ubiquitinated RTKs are recognized by components of the ESCRT (endosomal sorting complex required for
11
transport) machinery, which recruit receptors onto a flat bilayered clathrin lattice on the sorting endosome.
12
RTKs are subsequently sorted for internalization into the endosomal lumen. Non-ubiquitinated RTKs are not
13
efficiently recruited for internalization and can remain on the endosomal membrane as active signaling
14
molecules. Endosomes that become filled with intralumenal vesicles containing RTKs, known as multivesicular
15
bodies (MVBs), fuse with lysosomes to degrade the lumenal contents. Chimeric RTKs, which result from
16
chromosomal translocations, are no longer targeted to the plasma membrane, precluding them from down-
17
regulation through the endocytic pathway. If these chimeric fusion proteins are ubiquitinated (mono or K63),
18
they are not recognized by components of the 26S proteasome for degradation. Cbl may be sequestered from
19
mediating RTK ubiquitination by proteins such as Src, Sprouty2, cortactin, E5 and β-Pix. Proteins of the
20
endocytic pathway written in orange have been identified in human cancers as being mutated, over or under-
21
expressed or are frequent substrates for chromosomal translocations. Endophillin II is recruited to the plasma
22
membrane by CIN85 and induces negative membrane curvature to produce CCPs (130, 163). HIP1 (Huntington
23
interacting protein 1), CALM/Ap180 (clathrin assembly myeloid lymphoid leukemia), Numb are clathrin coat
24
adaptor molecules. Rabaptin5 is a Rab5 GAP involved in regulating endosome fusion (164) and has been found
25
as a chromosomal translocation product with the PDGFRβ (100). Hrs (Hepatocyte Growth Factor regulated
26
tyrosine kinase substrate), STAM (Signal Transducing Adaptor Molecule), Eps15, Eps15b is the isoform, which
27
functions in ESCRT0 (145), HCRP1 (Hepatocellular Carcinoma Related Protein 1) and TSG101 (Tumor
28
Susceptibility Gene 101).
29
15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
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Abella and Park. Figure 1 CCP P
U
P
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P
Src
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CIN85 P
Cbl
X
U
U
U
P
P
AP2 Eps15 HIP1 Numb CALM
P
P
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P
P
U
U
U
P
P
U
Kinase Kinase
Dimer Dimer
CCV
X
P
Cbl
Endophilin II
β-Pix Sprouty2 Cortactin
Cbl
U
P
P
P
Proteasomal Degradation
Sorting endosome
Early endosome P
U
Rabaptin 5
Growth Factor
wt RTK P
P
U U
P
Kinase Kinase
Dimer Dimer
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U
Chimeric RTK
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Clathrin coat
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RTK uncoupled from Cbl
MVB U
Ubiquitin
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Phospho-tyrosine Bilayered Clathrin Lattice
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Hrs STAM Eps15b
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