Ubiquitination-Dependent Regulation of Signaling Receptors in Cancer

Introduction

In a multicellular organism, functions of individual cells are dynamically regulated to ensure their cooperation with other cells to enable a collective tissue and organ function. Most of this concerted regulation is mediated by endocrine, paracrine, or autocrine effects of cell-produced soluble or cell surface/matrix–associated factors such as hormones, growth factors, cytokines, or other ligands of a peptide nature. Upon interaction with their cognate receptors on the surface of target cells, these factors trigger a myriad of intracellular signaling events. As an outcome of these signal transduction pathways, specific changes in cell transcriptional programs occur. In turn, these changes lead to the induction of expression of genes whose products assist the cells to perform their functions within the tissue and enable these cells to cope with environmental challenges.

The success or failure in realization of these programs is universally decided by the sensitivity of cells to the above-mentioned peptide regulators. This sensitivity is largely determined by the levels of a specific signaling receptor on the cell surface. The latter rely on receptor synthesis, maturation, and delivery to the plasma membrane, as well as on receptor degradation. The regulatory mechanisms that enable specific, rapid, and often irreversible changes in cell sensitivity to peptide ligand have largely evolved toward control of the receptor downregulation and degradation. These processes are often regulated by posttranslational modifications of the receptors including their phosphorylation and ubiquitination. Ubiquitination (along with the branch of signaling that enables ubiquitination, termed eliminative signaling) represents a universal and major mode by which the receptor downregulation may occur. ¹

As each signaling pathway has a component of negative regulation, this very component can be used or opposed by cancer cells depending on the physiological outcomes triggered by specific ligands. Cancer is often referred to as a disease of disregulated signaling. Indeed, abnormal sensitivity or refractoriness of individual cells to regulatory peptides lies in the center of tissue homeostasis abnormalities that are characteristics of neoplastic growth. ² A great deal of progress has been recently achieved in molecular characterization of alterations in the signaling networks that contribute to the pathogenesis of cancer. These new studies further highlight the role of signaling receptors and regulators of their levels in malignant transformation of cells and tumor development and progression and, to various extents, pave the road to a better treatment. ^3-5

Cancer cells often employ diverse strategies that are aimed at changing the levels of signaling receptors in order to increase the outcome of signaling conducive to cell growth and survival while decreasing the efficacy of those pathways that curb cell replication and induce cell death (Fig. 1). These strategies may include alterations in receptor phosphorylation and ubiquitination that either promote or inhibit the efficacy of receptor downregulation and degradation. An ever growing list of experimental evidence appears to directly or indirectly illustrate this synthetic paradigm. These new and exciting developments have prompted us to summarize available evidence and analyze several most pertinent examples in this review. We aimed to highlight common mechanisms by which cancer cells modulate either signaling that leads to ubiquitination or ubiquitination per se to affect the rate of downregulation of signaling receptors. We further discuss how these alterations affect the sensitivity of cells to factors that modulate their growth and survival. A better understanding of these mechanisms is required for advances in delineating the pathogenesis of human and animal cancers. Importantly, the enzymatic nature of key regulators (protein kinases or protein ubiquitin ligases) that are altered in cancer cells to achieve growth and survival advantages raises a certain level of hope that they could be targeted for successful therapy.

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Figure 1.

Alterations of signaling pathways and signaling receptors in cancers. Precancerous and tumor cells are bombarded by stimuli, the responses to which are either conducive (ligand A) or prohibitive (ligand B) for tumor development. Activation of cognate receptors (A1 or B1) leads to downstream stimulation of signaling elements (A2/B2) and executing transcription factors (A3/B3) that carry out functional changes in respective transcriptional programs. The ability of cells to respond to both types of stimuli is largely determined by the levels of receptors (A1 and B1) on the cell surface. Both types of receptors undergo internalization and postinternalization sorting into the late endosomes/lysosomes. The latter step irreversibly eliminates the ability of receptors to signal (depicted by a star) and leads to receptor degradation. Signaling elements downstream of receptors may contribute to receptor downregulation either at the stage of internalization (as shown for A1 as an example) or postinternalization sorting (as shown for B1) or both. Common alterations of receptors found in cancer lead to stabilization of receptors that signal to increase cell proliferation and viability (e.g., A1) and to accelerate the degradation of receptors that signal towards growth inhibition and cell death (e.g., B1).

Role of Ubiquitination in Receptor Endocytosis, Degradation in the Biosynthetic Pathway, and via Autophagy

Signaling receptors undergo endocytosis that removes them from the cell surface. This process by itself limits the sensitivity of a cell to a ligand that it may encounter in the future. An internalized receptor may then be recycled back to the cell surface or be targeted toward late endosomes for subsequent lysosomal degradation (reviewed in Acconcia et al. ⁶ ).

These processes can occur at a rather slow rate of nonspecific basal plasma membrane turnover, or they can be dramatically accelerated by signaling events induced either by specific ligands or inducers or various cross-signaling events. Ubiquitination of signaling receptors or their adaptors has been established as a posttranslational modification of paramount importance that mediates an increase in the efficacy of either internalization or postinternalization sorting to the lysosomes (and subsequent lysosomal degradation) or both. Several excellent reviews on this subject have been written to illuminate the paradigmatic views on the mechanisms by which ubiquitination affects the endocytic rate of various receptors. ^6-9 Here, we want to focus on some mechanistic elements of the ubiquitination-stimulated receptor endocytosis that could be used by cancer cells to either accelerate or slow down the entire process.

Besides controlling the endocytic rates, ubiquitination of the receptor precursor remains an important factor in the regulation of receptor stability within the biosynthetic pathway. Cell surface receptors undergo a complicated process of maturation and delivery to the plasma membrane (reviewed in Schnell and Hebert ¹⁰ ). During this process, numerous receptors become the client proteins for cellular chaperones such as Hsp90 and Hsc70 and can be ubiquitinated and degraded by 26S proteasomes in a manner dependent on a chaperone-dependent E3 ubiquitin ligase termed “carboxyl terminus of Hsc70-interacting protein” (CHIP) (reviewed in Takahashi and Imai ¹¹ ). This regulation will plausibly affect the overall levels of the receptor and, consequently, an ability of a cell to furnish its plasma membranes with a number of receptors that is sufficient for sensing a regulatory signal from a given ligand. The rate of chaperone- and CHIP-dependent ubiquitination and degradation of client receptors can be affected by specific chaperone inhibitors and is often used in anticancer therapy. ¹²

In addition, ubiquitination plays an important role in autophagy, a mostly prosurvival lysosome-centered mechanism of cellular self-digestion that is often activated by cells under conditions of nutrient deprivation or exposure to anticancer drugs. ^13-15 Ubiquitination regulates not only the formation of vacuoles to be fused with lysosomes but also potentially the selection of cargo receptors for these vacuoles. ^16,17 These mechanisms could contribute to the regulation of a number of signaling receptors and, accordingly, cell sensitivity to important growth factors and cytokines under such conditions. ^18,19

Altering Receptor Ubiquitination: Changes in Ubiquitin Ligases or Affinity of Receptors to These Ligases

How is receptor ubiquitination regulated, and how can this regulation be altered in cancer cells to suit their progrowth and prosurvival agenda? The rate of ubiquitin conjugation to a receptor is determined by 2 distinctive factors including the ability of the intracellular domain of a receptor to recruit a specific E3 ubiquitin ligase and on the overall activity of such a ligase (Fig. 2). The latter factor is a sum of such E3 ligase expression levels and catalytic activity. This factor is of paramount importance; examples provided in the next section will highlight how the modulation of overall activity of a given E3 ubiquitin ligase can alter the ubiquitination state of a receptor and, accordingly, accelerate or retard its degradation.

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Figure 2.

Mechanisms underlying signal-stimulated increase in receptor ubiquitination. Activation of signaling receptors usually promotes ubiquitination of these receptors. An increase in receptor ubiquitination and subsequent degradation can occur due to overall activation of E3 (left side) and/or increased recruitment of E3 to the substrate (right side). The former includes an increase in expression of the respective E3 and/or its activity; the latter is often mediated by additional posttranslational modifications of the receptor (e.g., phosphorylation). Both pathways are often exploited by cancer cells to modify ubiquitination and stability of signaling receptors.

Another mode of regulation is focused on the recruitment of a specific E3 ubiquitin ligase to the receptor. An increased (or decreased) ability of an intracellular domain of a receptor to interact with its ubiquitin ligases is expected to accelerate or slow down the rate of its ubiquitination and degradation. These alterations could occur as a result of direct mutation of the receptor that could happen in a cancer cell. In addition, alterations in the affinity of signaling receptors to E3 ligases may result from other somatic mutations that change the outcome of signaling leading to E3 recruitment. ⁷

In the context of a ligand-inducible signal transduction pathway, ligase recruitment is often stimulated by a secondary posttranslational modification (e.g., phosphorylation) of the receptor’s intracellular domain. These modifications increase the affinity of the receptor to its E3 enzyme and, accordingly, enable efficient ubiquitination of the receptor. For example, phosphorylation of epidermal growth factor receptor on specific tyrosine residues mediated by activated EGFR kinase enables the recruitment of its E3 ligase, c-Cbl. ²⁰

Posttranslational modifications that increase receptor affinity to an E3 ligase are usually mediated by enzymes that are activated by a branch of the main signal transduction pathway that actually activates the receptor (Fig. 3). This “branching off” can occur at the level of a specific acceptor of a given modification such as Tyr phosphorylation of EGFR and other RTKs; it both signals forward and enables the recruitment of c-Cbl for downregulation. Alternatively, there could be an activation of additional enzymes that specifically act to promote ubiquitination of a receptor and eliminate its ability to further contribute to the cellular responses to its ligand (as for Janus kinase-dependent serine phosphorylation of some cytokine receptors, see below). It is also plausible that this “eliminative signaling” could be inducible not only by the receptor’s own ligands but also by heterologous cross-acting ligands or other physiological/pathological process inducers that activate a similar pathway. Furthermore, a pathological, constitutively active signaling process might be capable of furnishing the receptors with modifications that would increase their ability to recruit the respective E3 ligase. Using examples from studies carried out by our laboratory and other groups, we will discuss how cancer-associated alterations of eliminative signaling can affect the rate of downregulation and degradation of signaling receptors in tumor cells in the next section.

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Figure 3.

Mechanisms underlying the regulation of signaling pathways. Cellular responses to a specific ligand (e.g., ligand A) result in changes of the gene expression program. Some of the products of ligand A–inducible genes might be capable of inhibiting the signaling cascade via negative feedback (exemplified by cytokine-mediated induction of tyrosine phosphatases of JAK inhibitors and denoted by a blue solid arrow). However, a much more rapid regulation of signaling can occur due to the branching of signaling pathways. In this example, “stem” signaling induced by ligand A includes activation of receptor A1 followed by activation of a signaling element A2. Subsequent “forward” signaling involves activation of A3 and other elements that lead to transcription changes. Concurrently, eliminative signaling (involving A2-dependent activation of a factor X and effect of X on receptor downregulation denoted by a solid red arrow) results in rapid downregulation and degradation of A1, thereby restricting the duration and magnitude of cellular responses to ligand A. This pathway could be employed by an unrelated signaling pathway via “cross”-signaling initiated by ligand B, which can lower the sensitivity of the cell to ligand A via activation of factor X.

Alterations of Ubiquitin-Dependent Regulation of Signaling Receptors in Cancers

Cancer cells evolve to promote their own growth and to withstand antiproliferative and antisurvival stimuli. Diverse strategies that enable this evolution often include alterations in ubiquitination and degradation of signaling receptors. Such alterations include stabilization of progrowth/prosurvival receptors as well as accelerated degradation of those receptors whose downstream signaling is detrimental for cell proliferation and viability. Molecular mechanisms underlying these changes include changes in levels and activities of specific E3 ubiquitin ligases or in an adjusted affinity of receptor substrates to these ligases. The latter, in turn, can occur due to either posttranslational receptor modifications or direct mutations in the receptors.

Stabilization of Receptors that Signal to Promote Cell Growth and Survival

Impeded ubiquitin-mediated degradation of receptors that mediate signaling induced by growth factors and prosurvival ligands often occurs in various human cancers. These events have been described by many research groups that have studied mechanisms of downregulation of receptor tyrosine kinases (RTKs) and of cytokine/hormone receptors.

EGFR

The epidermal growth factor receptor (EGFR) is activated by the growth factors such as EGF or transforming growth factor–α. Extracellular interaction with a ligand results in homodimerization of EGFR and ensuing activation of its tyrosine kinase activity and phosphorylation of the tyrosine residues within the intracellular domain. The latter enables the recruitment of downstream effectors and activation of cell survival and proliferating signals involving the PI3K-Akt and MAPK pathway. ^21,22

Activation of EGFR not only engages these pathways but also triggers its own downregulation and degradation. This process depends on the recruit-ment of the c-Cbl (for Casitas B–lineage lymphoma protein) E3 ubiquitin ligase to specific phosphorylated tyrosine residues within the intracellular domain of EGFR. This domain then becomes ubiquitinated with the aid of ubiquitin-conjugating enzymes Ubc4 and Ubc5. ²³ Whereas the role of EGFR ubiquitination in internalization of the receptor is not entirely clear, it has been unequivocally demonstrated that ubiquitination prevents EGFR recycling and promotes targeting of already internalized EGFR molecules toward the lysosomes where EGFR degradation occurs. These events eliminate all branches of signaling initiated by EGF and limit the magnitude and duration of progrowth and prosurvival responses (reviewed in Sorkin and Goh ²⁰ and Madshus and Stang ²⁴ ).

Importance of this regulation for malignant cell transformation became evident upon demonstration that c-Cbl, a known regulator of EGF signaling, contains the RING domain (required to recruit ubiquitin-conjugating enzymes) and functions as an EGFR E3 ubiquitin ligase. ^25,26 Previous studies on a product of retroviral oncogenes, v-Cbl, have shown that this protein, which was capable of transforming mammalian cells in vitro, turned out to be a truncated form of c-Cbl that lacked the RING domain. It appears that v-Cbl acts as a dominant-negative mutant that competes with endogenous c-Cbl and therefore blocks the c-Cbl–mediated negative regulation of RTKs (reviewed in Thien and Langdon ²⁷ ).

To function as an efficient E3 ligase for EGFR (and some other RTKs, see below), c-Cbl requires not only the RING domain but also the tyrosine kinase–binding domain and an intact α-helical linker that properly positions the substrate toward ubiquitin-conjugating enzymes. Mutations in either of these domains turn c-Cbl into a highly oncogenic mutant that prevents RTK downregulation and rapidly transforms mammalian cells. ²⁸ Conversely, c-Cbl overexpression has been shown to promote EGFR ubiquitination and downregulation and to limit the extent of growth factor signaling. ²⁹

EGFR belongs to the ErbB family, which also includes Her2/Neu/ErbB2, ErbB3, and ErbB4. These receptors, whose stability is regulated similarly to EGFR, have been linked to a number of human cancers, including lung and breast cancers, squamous carcinomas, gliomas, and others (reviewed in Mani and Gelmann ³⁰ and Sebastian et al. ³¹ ). Dysregulated constitutive activation of EGFR signaling in human cancers occurs via multiple mechanisms such as receptor overexpression, activating mutations, and/or impaired turnover. ³¹ As a result, EGFR is activated and in turn influences tumor cell survival, proliferation, angiogenesis, and invasiveness.

Human cancer cells have developed diverse strategies to decrease ubiquitination and downregulation of EGFR by c-Cbl. For example, a naturally occurring deletion mutant of EGFR that lacks exons 2 to 7 has been reported as the most common EGFR mutation in diverse types of human tumors including glioma, ³² glioblastoma, ³³ bladder, ³⁴ and lung cancers. ³⁵ This deletion mutation results in a EGFR missing a large part of the extracellular ligand-binding domain. ³⁶ This mutation impedes the ability of activated EGFR to recruit c-Cbl. ³⁷ As a result, in the absence of c-Cbl–mediated ubiquitination, EGFR becomes stable and constitutively active. ^38,39

Another class of clinically important mutations of EGFR is formed by the mutations in the tyrosine kinase domain that occur in a subset of patients with lung cancer showing a dramatic response to EGFR tyrosine kinase inhibitors such as gefitinib. ^40,41 However, initial success in therapy using these inhibitors is often followed by resistance. Investigation of gefitinib-resistant EGFR mutants that spontaneously arise in non–small cell lung cancers revealed that some of these mutants are refractory to EGF-induced ubiquitination and downregulation and, as a result, display a greater extent of signaling. ⁴²

Yet another mechanism by which cancers stabilize EGFR is high levels of expression of its related receptor Neu/Her2/ErbB2. Overexpression of ErbB2 (reported in breast, ovary, and prostate cancers) favors the formation of EGFR/ ErbB2 heterodimers, which display a lesser ability to recruit c-Cbl compared to EGFR homodimers. Impaired degradation of these heterodimers contributes to elevated signaling and ensuing advantages in growth and survival of these tumors. ⁴³

Met

The Met protein is a high-affinity receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF). In response to HGF/SF stimulation, Met RTK was multimerized and autophosphorylated at specific tyrosine residues within its intracellular region. Activated Met then recruits downstream adaptor proteins such as Gab-1, Grb2, Shc, and c-Cbl and subsequently activates PI3K/Akt, PLC-γ, STATs, and Ras/Raf/MEK/ERK, which are important for cell proliferation, cell movement, and morphogenic differentiation. ⁴⁴

Similar to other RTKs, Met is degraded in a ubiquitination-dependent manner. ⁴⁵ This ubiquitination process is mediated by c-Cbl E3 ubiquitin ligase that binds to Met at a juxtamembrane tyrosine residue via its tyrosine kinase–binding domain. Ubiquitinated Met then undergoes endocytosis and is transported to the late endosomal compartment, where it is ultimately degraded. Importantly, an oncogenic Met mutant was isolated during a chemical mutagenesis screen. This mutant (Tpr-Met) exhibited greatly increased signaling and the ability to transform mammalian fibroblasts and epithelial cells. ⁴⁶ Subsequent analysis revealed that an increase in transforming the oncogenic activity of Tpr-MET stems from the deletion of juxtamembrane tyrosine residue and resulting failure to recruit c-Cbl. This failure, in turn, leads to impaired ubiquitination and degradation as well as to constitutive activation of Met. ⁴⁷

Whereas these studies suggested that impaired Met degradation and hyperactivation may cause malignant transformation and tumorigenesis, an increased Met expression has been indeed detected in many human cancers. Moreover, Met overexpression has been associated with metastasis and poor prognosis. ⁴⁸ Although the mechanisms underlying Met dysregulation are diverse, ⁴⁹ there is evidence that stabilization of the Met protein is a contributing factor. For example, a subset of lung cancer patients was found to harbor the somatic intronic mutations of the Met kinase that lead to an alternatively spliced transcript. This transcript encodes a deletion of the juxtamembrane domain, resulting in the loss of c-Cbl binding. The mutant receptor exhibits decreased ubiquitination and delayed downregulation as well as constitutive high signaling in vitro. Furthermore, histopathological analysis shows a strong correlation of this mutation with an elevated Met protein expression and activation of the MAPK pathway in primary tumors. Cells expressing ubiquitination-refractory Met also exhibit robust ligand-mediated proliferation and significant in vivo tumor growth. ⁵⁰

Platelet-Derived Growth Factor Receptor

Platelet-derived growth factor receptor (PDGFR) family members are known to mediate proliferation, survival, and motility of connective tissue cells. ⁵¹ PDGF stimulation leads to the activation of intracellular signaling pathways via recruiting SH2 domain–containing signaling molecules, such as Src and PI3K, to specific phospho-tyrosine residues. As a result, cells can undergo proliferation, survival, and migration. ⁵²

Similar to EGFR and Met, PDGFR is also a substrate for c-Cbl. ^27,53 This E3 ubiquitin ligase associates with PDGFR upon its tyrosine phosphorylation. Resulting ubiquitination of PDGFR leads to receptor degradation and restricts PDGF signaling. Negative regulation elicited by endogenous c-Cbl decreases PDGF-induced cell proliferation and protection against apoptosis, ⁵⁴ whereas expression of oncogenic c-Cbl mutants upregulates PDGFR signaling, promotes cell transformation, and potentially contributes to tumorigenicity. ⁵⁵

Dysregulation and activation of PDGF signaling have been linked to tumorigenesis since discovery of the transforming retroviral v-sis oncogene derived from the PDGF B chain. In cancer patients, PDGFR can drive tumor growth by PDGF stimulation or by activating mutation. For instance, translocation of the PDGF B gene to the COL1A1 gene in dermatofibrosarcoma protuberans leads to autocrine PDGF stimulation. In gastrointestinal stromal tumors, there are mutations in the PDGFRα gene that unleash receptor kinase activity. In chronic myelomonocytic leukemia, fusion of the PDGFRβ gene with the TEL gene results in constitutive dimerization and activation of the intracellular receptor domain (reviewed in Ostman et al. ⁵¹ and Pietras et al. ⁵² ). The role of these mutational events in the stability of PDGFR and the extent to which stabilization of PDGFR contributes to its tumorigenicity remain to be investigated.

PRLr

Prolactin receptor (PRLr) is a class I cytokine receptor that is activated by lactogenic cytokines/hormones such as prolactin (PRL), placental lactogens, and growth hormone. ^56-58 Activation of PRLr leads to its dimerization and ensuing stimulation of diverse signaling pathways including Janus kinase (JAK) signal transducer and activator of the transcription (STAT) pathway as well as the MAPK, Src, and PI3K pathways. ⁵⁹ PRL produced by both pituitary gland and mammary epithelium regulates growth survival and differentiation of numerous cell types and plays a key role in mammary gland development. Emerging epidemiological and laboratory evidence implicates PRLr in breast tumorigenesis. ⁶⁰

Besides activating downstream signaling pathways, PRL stimulation also induces eliminative signaling that limits the extent of cellular responses to this hormone. PRL stimulates ubiquitination and degradation of PRLr via 2 diverse mechanisms that prevail in various cell types. One of these mechanisms relies on the activation of Src, which promotes internalization and degradation of PRLr through the clathrin-independent pathway. ^61,62 The second pathway involves the activation of JAK2 and a yet to be identified downstream Ser kinase. ⁶³ This kinase phosphorylates the PRLr intracellular domain on Ser349, leading to the recruitment of beta-transducin repeats-containing protein-2 (βTrcp2). This protein recruits the Skp–Cul–F-box (SCF) complex to form a multicomponent E3 ubiquitin ligase (SCF^βTrCP). ⁶⁴ The above-mentioned E3 ligase facilitates PRLr ubiquitination and subsequent clathrin-dependent endocytosis, postinternalization sorting, and lysosomal degradation. ^63,65,66

Interestingly, in cells that are deprived from mitogenic stimulation, an efficient basal Ser349 phosphorylation of PRLr (as well as its ubiquitination and degradation) can be mediated by constitutively active glycogen synthase kinase 3β (GSK3β). ⁶⁷ Inhibition of GSK3β by constitutive mitogenic and prosurvival signaling mediated by the MAPK and PI3K pathways leads to an impaired Ser349 phosphorylation of PRLr ⁶⁷ and a rather common stabilization of PRLr in primary human breast cancers. ⁶⁸ Conversely, human mammary epithelial cells expressing the PRLr mutant lacking Ser349 exhibit augmented PRL signaling, a greater proliferation rate, and an increased ability for invasion. ⁶⁹ Naturally occurring PRLr mutants that exhibit constitutive signaling have been recently identified in some breast cancers and in benign mammary tumors ^70,71 ; it remains to be investigated whether these mutants display altered rates of degradation.

Increased Degradation of Receptors that Mediate Anti–Growth/Survival Signaling

TRAIL Receptors

The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells when binding to its surface receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). DR4 and DR5 belong to the tumor necrosis factor (TNF) family of receptors, which also includes TNF-R1, FAS, and NGFR (reviewed in Locksley ⁸⁷ and Ozören and El-Deiry ⁸⁸ ). Apoptotic responses initiated upon DR4/5 stimulation are dependent on the catalytic function of caspases and may involve extrinsic (DISC–caspase 8–caspase 3) and intrinsic (Bid–cytochrome C release–Apaf–caspase 9–caspase 3) cascades. These effects of TRAIL could be prevented by expression of the TRAIL decoy receptors DcR1, DcR2, and OPG. ^89,90 In general, normal cells are resistant to TRAIL in part due to the expression of decoy receptors. ⁸⁸ Kohlhaas et al. reported that TRAIL and its receptors are rapidly endo-cytosed in a time- and concentration-dependent manner. Furthermore, inhibition of internalization of TRAIL robustly augments TRAIL-induced apoptosis. ⁹¹ The specifics of signaling and posttranslational events involved in this regulation (phosphorylation, ubiquitination, etc.) are under investigation. A first step in this direction was a recent report implicating c-Cbl in this process ⁹² ; however, independent verification of the role of c-Cbl and indication of how it gets recruited to TRAIL receptors remain to be elucidated.

TRAIL receptors are attractive targets for cancer therapy because they robustly kill cancer cells while sparing their nonmalignant counterparts. However, TRAIL resistance observed in some cancer cells was shown to limit the therapeutic potential. This resistance to TRAIL may occur by diverse mechanisms including inactivating mutations of DR5, overexpression of decoy receptors, or loss of DR4. ^88,89,93 Importantly, emerging evidence suggests that accelerated downregulation of DR4/5 may underlie some cases of resistance to TRAIL. Some breast cancer cell lines exhibit an accelerated rate of DR4 and DR5 endocytosis, which is detected even in the absence of TRAIL. ⁹⁴ Delineation of phosphorylation and ubiquitination events underlying this phenomenon is expected to enable the development of specific means that would slow down DR4/5 endocytosis and sensitize cells to cell death induced by TRAIL.

TGFβR

Transforming growth factor β (TGFβ) transduces its signals via heteromeric complexes of transmembrane serine-threonine kinase receptors involved in diverse developmental processes and pathogenesis of many diseases. Within the receptor complex, the TGFβ type II receptor (TβRII) is constitutively active and phosphorylates the TGFβ type I receptor (TβRI) on serine-threonine residues in response to TGFβ (reviewed in Ikushima and Miyazono ⁹⁵ ). Activated TβRI phosphorylates its downstream signaling mediators/effectors Smad2/3 and Smad4 ⁹⁶ and activates Smad-independent cascades of kinases including ERK, JNK, and p38 MAP kinase. ⁹⁷ In addition, activation of TFGβR activates eliminative signaling that leads to receptor downregulation and degradation and that limits cellular responses to TGFβ. Recent reports show that TGFβ receptors can be internalized and downregulated via a ubiquitin-mediated mechanism as a way of signaling modulation. ⁹⁸ TβRI downregulation is mediated by E3 ligase Smad-ubiquitination regulatory factor 1 (Smurf1), ^98,99 while TβRII downregulation is mediated by E3 ligase Smad-ubiquitination regulatory factor 2 (Smurf2); both belong to the HECT class of ubiquitin ligases. ¹⁰⁰ Mechanisms underlying the recruitment of Smurf1/2 to the receptor involve additional negative regulator Smad7 ¹⁰¹ ; posttranslational events that contribute to these interactions are largely to be characterized.

In most cell types, TGFβ can inhibit cell growth, for example, via cell-cycle progression by transcriptional repression of the growth promoting gene Myc. ¹⁰² Hence, aberration of TGFβ signaling can promote tumor growth. It has been reported that high-level expression of the Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma. ¹⁰³ In addition, overexpression of Smurf2 promotes metastasis and increases migration and invasiveness of breast cancer cells. ¹⁰⁴ Posttranscriptional downregulation of TβRII was frequently found in tissues of renal cell carcinoma compared to normal renal tissues. It has also been shown that the level of Smurf2 was increased in renal cell carcinoma tissues that inversely correlated to their TβRII levels. ¹⁰⁵ Using a genome-wide approach, it was identified that Smurf1 is amplified in pancreatic cancer. ^106,107 In addition, RING finger protein 11 was shown to be overexpressed in breast cancers. ¹⁰⁸ This protein was also shown to interact with Smurf2 and antagonize its effects directed at ubiquitination and degradation of the TGFβ receptor and inhibition of TGFβ signaling. ¹⁰⁹ Moreover, ubiquitin regulates TGFβ signaling in cancers not only on the receptor level but also on various levels of downstream Smad proteins. ¹¹⁰

Implications for Anticancer Therapy

Exploitation of protein ubiquitination and degradation in general represents a novel and promising trend in the development of targeted anticancer therapies. ¹¹¹ This trend includes the development of drugs that would inhibit degradation of anti–growth/survival receptors (such as DR4/5 or IFNAR1) or promote the degradation of receptors whose signaling is conducive for the proliferation and survival of cancer cells (such as EGFR, MET, or PRLr). Some of these directions that have been already developed by design or serendipity are highlighted below.

Given that levels and activities of β-Trcp E3 ubiquitin ligase are maintained by signaling downstream of the activated BRAF mutant, an effort to stabilize IFNAR1 and to augment the therapeutic efficacy of IFNα in malignant melanomas has been proposed. Although IFNAR1 stabilization was indeed achieved in melanoma cells treated with the Raf inhibitor sorafenib, this agent failed to augment the antigrowth effects of IFNα, most likely due to peripheral suppression of JAK. ⁸⁶ The development of novel and selective Raf inhibitors that attenuate β-Trcp–mediated ubiquitination yet spare JAK activity is expected to improve the efficacy of IFNα therapy. Additional efforts toward inhibiting the phosphorylation of IFNAR1 that enables β-Trcp recruitment are also warranted.

The antitumor efficacy of Her2/Neu/ErbB2-specific antibodies such as Herceptin (Genentech Inc., South San Francisco, CA) is dependent on the ability of these agents to stimulate Cbl-dependent ubiquitination and of Her2 leading to Her2 degradation. ^112-114 It is plausible that a similar role of ubiquitination might be revealed in the mechanisms of action of other antireceptor agents targeted against other c-Cbl substrates (e.g., MET, PDGFR, etc.).

Constitutively active mutants of the signaling receptor FLT-3 (FMS-like tyrosine kinase 3) play a central role in the pathogenesis of some cases of acute or chronic acute myeloid leukemia. Acceleration of ubiquitination and degradation of FLT-3 at least in part contribute to the antitumorigenic effect of geldanamycin and its derivatives that are potent inhibitors of the Hsp90 chaperone. ¹¹⁵ Similar stimulation of ubiquitination by inhibiting Hsp90 has been reported in regard to other signaling receptors including Neu/Her2 /ErbB2 ^116,117 and RON, the tyrosine kinase receptor for macrophage-stimulating protein, which becomes oncogenic upon a point mutation in the kinase domain. ¹¹⁸

Some existing therapeutics or promising agents have been found to promote the downregulation of pro–growth/ survival receptors. For example, endogenous cannabinoid anandamide was shown to accelerate the downregulation of PRLr ¹¹⁹ via induction of Ser349 phosphorylation independently of the activity of GSK3β. ⁶⁹ Conversely, a novel antagonist of type I cAMP-dependent protein kinase was shown to promote the downregulation of EGFR. ¹²⁰ Intriguingly, the treatment of head and neck cancer cells with cisplatin robustly accelerated the degradation of EGFR; this degradation was of importance in mediating the effects of cisplatin in these cancers. ¹²¹ Similarly, accelerated EGFR degradation is central for the chemopreventive effects of some natural compounds contained in green tea. ¹²² Delineation of mechanisms underlying these cross-signaling regulatory events is expected to bring out novel targets for efficient anticancer therapy.

Conclusions

Signaling receptors located at the cell surface determine the sensitivity of cells to collective regulation of tissue function, proliferation, and survival. Egoistic behavior of cancer cells is often mediated by alterations in the availability of these receptors and resulting augmentation of pro–growth/survival signaling and attenuation of cellular responses to negative regulators. Whereas many of these processes contribute to the pathogenesis of human cancers, mechanisms underlying these alterations could be also exploited for anticancer treatment.