The regulatory crosstalk between kinases and proteases in ... - Nature

REVIEWS

The regulatory crosstalk between kinases and proteases in cancer Carlos López-Otín* and Tony Hunter‡

Abstract | Kinases and proteases are responsible for two fundamental regulatory mechanisms — phosphorylation and proteolysis — that orchestrate the rhythms of life and death in all organisms. Recent studies have highlighted the elaborate interplay between both post-translational regulatory systems. Many intracellular or pericellular proteases are regulated by phosphorylation, whereas multiple kinases are activated or inactivated by proteolytic cleavage. The functional consequences of this regulatory crosstalk are especially relevant in the different stages of cancer progression. What are the clinical implications derived from the fertile dialogue between kinases and proteases in cancer? Proteasome Multimeric and multicatalytic protease that degrades multiple intracellular proteins in a ubiquitin- and ATP-dependent manner and has a key role in many events of cellular regulation mediated by protein degradation.

*Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain and ‡ Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, CA 92037, USA. Correspondence to C. L.-O. e-mail: [email protected] doi:10.1038/nrc2823 Published online 19 March 2010

Kinases and proteases have essential roles in many biological and pathological processes, including cancer1,2. The reactions catalysed by these enzymes are fundamentally different: phosphorylation is physiologically reversible but proteolysis is essentially irreversible. The irreversibility of protease-catalysed reactions and the classic idea that proteases were nonspecific demolition agents associated with protein catabolism meant that these enzymes were overlooked as important biological regulators. However, we now recognize that proteases carry out highly selective cleavage of specific substrates2. This realization has resulted in the establishment of many parallels between kinases and proteases. Human and model organism genome sequences have revealed that the enormous diversity in kinase and protease functions derives from the evolution of many distinct enzymes with the ability to catalyse phosphorylation reactions and hydrolysis of peptide bonds, respectively 3,4 (BOX 1) . The human kinome — the complete set of kinases produced by human cells — consists of at least 518 components distributed into 10 different groups3 (FIG. 1). The dimension of the human degradome — the complete set of human protease genes — is similar, consisting of at least 569 components distributed into 5 catalytic classes5 (FIG. 1). Moreover, the structures of both enzyme families have revealed that their catalytic domains are commonly linked to various specialized modules that influence kinetic properties, substrate specificity and cellular localization. Therefore, an emerging theme in the fields of kinases and proteases is one of diversity and multiplicity, which has created additional options to regulate biological processes. In addition, more than 100 of the

569 proteases are regulated by phosphorylation, and more than 100 of the 518 kinases are targeted by specific proteases (FIG. 1; see Supplementary information S1,S2 (tables)), underscoring the extensive crosstalk between these enzymes. In this Review we discuss the direct functional interactions between kinases and proteases in both normal and tumour tissues and the potential clinical applications derived from the recognition of the interplay between these complex enzymatic systems.

Direct kinase–protease interactions Phosphorylation by protein kinases can switch protease activity on or off. Among the growing list of human proteases that are activated by kinases (Supplementary information S1 (table)) are the metalloproteinases, such as those of the a disintegrin and metalloproteinase (ADAM) family, cysteine proteases such as calpains, serine proteases such as OMI (also known as HTRA2), and threonine proteases including diverse proteasome subunits6–9. By contrast, some proteases are inactivated after phosphorylation, as is the case for several caspases and separase10,11. Augmenting this primary level of kinase-mediated protease regulation, kinases can also modulate protease function through changes in half-life, subcellular localization and ability to interact with other molecules (FIG. 2). Proteases can also switch kinases on or off (Supplementary information S2 (table)). For example, calpains, caspases and granzyme B can remove inhibitory domains from kinases and lead to their activation7,12,13. By contrast, other proteolytic enzymes target different kinases and cause their degradation, as illustrated by caspase 3 inactivating focal adhesion kinase (FAK; also known as PTK2)

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REVIEWS At a glance • Protein kinases and proteases are both major classes of cellular enzymes; the kinome and the degradome each include more than 500 genes. • Direct interactions between kinases and proteases are frequent, and more than 125 examples of kinases that undergo regulated processing by one or more proteases are currently known. Targets for proteases include kinases in all the major branches of the kinome, as well as atypical protein kinases. Conversely, approximately 150 proteases in all the main families in the degradome are known to be phosphorylated by one or more kinases.

by proteolytic cleavage14. Additionally, proteases can modulate kinase functions in more complex ways, including the conversion of a receptor tyrosine kinase (RTK) ectodomain to a soluble protein or the generation of intracellular fragments with new signalling agendas15,16. These processes are well illustrated by the shedding and regulated intramembrane proteolysis ( RIPping ) events in which ADAMs and presenilins sequentially act on many RTKs and so modify their signalling properties6 (FIG. 2). This molecular dialogue between phosphorylation and proteolysis occurs in many physiological and pathological conditions, but is especially relevant for inducing cell proliferation, survival and invasion, which are crucial for cancer progression.

Proteasome. The close interdependence between CDK activity and the irreversible proteolysis that is mediated by the ubiquitin proteasome system (uPS) is essential for providing directionality to the cell cycle. Additionally, the phosphorylation of many substrate proteins is crucial for marking their degradation by the uPS, extending the links between both post-translational regulatory mechanisms. The bidirectional communication between protein kinases and the proteasome is especially evident from the finding that the activation of many kinases triggers their subsequent downregulation through the uPS pathway 19. Accordingly, activation-dependent degradation and signalling termination of non-receptor TKs, including SRC family kinases, ABl, FAK and Janus kinase 2 (JAK2), commonly occurs through uPSmediated mechanisms usually involving members of the CBl family of E3 ligases. likewise, non-receptor serine/threonine kinases such as protein kinase Cs (PKCs), MAPKs and AKT, and receptor serine/threonine kinases including transforming growth factor-β (TGFβ) receptors are also degraded through the uPS pathway 19. Additionally, activated kinases can in some cases be inactivated by the destruction of their activating regulators or the upregulation of their inhibitors17,19. The importance of direct and indirect mechanisms of kinase downregulation is emphasized by observations that their disruption results in sustained kinase signalling that can lead to cancer 19,20. The interplay between kinases and the proteasome is further enriched by evidence that the proteolytic activity of the proteasome is itself regulated by phosphorylation9. These kinase-mediated events contribute to the assembly of the intact 26S proteasome and result in the positive regulation of its activity 9,21. The discovery of proteasome subunit phosphorylation, together with the identification of kinases and phosphatases as associating partners of this proteolytic machine, supports the relevance of phosphorylation events as regulatory signals for the modulation of proteasome activity.

Kinase–protease interplay in cell proliferation The first indications that direct interactions between kinases and proteases could influence tumour development were derived from studies on the mechanisms of cell division. The finding that proteolysis drives cell cycle progression by regulating the activity of cyclindependent kinases (CDKs) was a landmark in this field and demonstrated that the cell cycle could no longer be considered as an exclusive kinase cycle17. Attention originally focused on proteolytic events involving the degradation of ubiquitylated substrates by the proteasome18. However, recent studies have revealed that other proteolytic machines such as the signalosome, complex proteolytic systems such as those involving deubiquitylases (DuBs) and individual proteases like separase also contribute to the regulation of cell cycle

Signalosome. The COP9 signalosome (CSN) consists of eight subunits, with CSN5 (also known as JAB1) responsible for CSN isopeptidase activity. The CSN is a molecular platform connecting kinase signalling and proteolysis through its ability to associate with different protein kinases, such as casein kinase 2 (CK2) and protein kinase D1 (PRKD1), which in turn phosphorylate several CSN subunits22. Further studies have shown that CSN5 induces the degradation of the CDK inhibitor p27 and facilitates cell cycle progression downstream of the BCR–ABl fusion kinase in human leukaemia cells23. The involvement of CSN5 in the regulation of cell proliferation is relevant to recent clinical findings of the deregulation of the CSN and its individual subunits — particularly CSN5 — in cancer progression24. The observation that downregulation of CSN5 in cultured cancer

• Bidirectional kinase–protease interactions have an important role in many cellular processes, including transmembrane signalling, apoptosis and cell migration. Prominent examples include kinase–caspase crosstalk in apoptosis and interactions between deubiquitylases or γ‑secretase and receptor tyrosine kinases in cell proliferation and invasion. • Kinase–protease interactions are implicated as causal events in disease, and have a particularly important role in cancer. Cell migration and invasion are hallmarks of metastatic cancer, and many kinase–protease interactions are involved in activating these processes in a dysregulated fashion in cancer cells. • Clinical implications of kinase–protease crosstalk are emerging. Interplay between ERBB family receptor tyrosine kinases and a disintegrin and metalloproteinase (ADAM) proteases results in the shedding of ERBB2 soluble forms that blunt the efficiency of ERBB2‑specific monoclonal antibody therapy in breast cancer. A clinical trial in patients with breast cancer is underway using a combination of an ADAM10 and ADAM17 inhibitor with an ERBB2‑specific antibody. • Kinase–protease connections are being uncovered at a rapid pace, and their roles in cancer will undoubtedly become increasingly important and offer new opportunities for clinical intervention.

Shedding Protease-mediated process that involves the release of the extracellular portion of a membrane protein.

RIPping Protease-mediated process in which a receptor is cleaved in the transmembrane domain by intramembrane-cleaving proteases (I-CLiPs), a group of proteases including the presenilins that are part of a larger complex called γ-secretase.

Signalosome Multi-subunit protease that promotes the activity of cullin-containing ubiquitin E3 ligases and has important roles in the cell cycle, DNA damage response, nuclear transport, gene transcription and kinase signalling.

Deubiquitylases A large family of enzymes that comprises cysteine proteases and metalloproteinases, which remove conjugated ubiquitin from proteins and are linked to the regulation and termination of ubiquitin-mediated signalling.

progression through specific bidirectional interactions with kinases. All these proteolytic systems or enzymes are directly regulated by phosphorylation and in turn target kinases for activation or inactivation.

NATuRe ReVIewS | CanCer

VOluMe 10 | APRIl 2010 | 279 © 2010 Macmillan Publishers Limited. All rights reserved

REVIEWS E3 ligase Enzyme that catalyses the final step of the process that activates and transfers ubiquitin and ubiquitin-like molecules to substrate proteins.

Isopeptidase Enzyme that cleaves amide bonds that are not present in the main polypeptide chain of a protein, such as those formed between the ε-amino group of lysine side chains of ubiquitin-targeted proteins and the terminal carboxyl group of ubiquitin.

cells reduces cellular proliferation23, together with the fact that CSN5 knock down in murine xenografts inhibits tumour growth25, suggests that CSN5 could be an attractive target for therapeutic intervention in cancer. DUBs. The DuBs are a large family of approximately 100 enzymes that are linked to the regulation and termination of ubiquitin-mediated signalling. They are classified into five groups according to structural and functional similarities: ubiquitin-specific proteases (uSPs), ubiquitin carboxy-terminal hydrolases (uCHs), ovarian-tumour proteases (OTus), Machado– Joseph disease protein domain proteases (MJDs) and JAB1/MPN domain-associated metallopeptidases (JAMMs)26. DuBs control cell proliferation through diverse mechanisms, including the regulation of cell cycle checkpoints, modulation of growth factor signalling and trafficking, remodelling of chromatin structure and protection of transcription factors from unscheduled degradation26. The relevance of DuBs in cell cycle progression is underscored by the fact that some family members are integral components of the core cell cycle machinery and cell cycle checkpoints. Functional analyses of DuBs have delineated their complex interactions with kinases in cell growth control (FIG. 3). uSP8 has an important role in RTK stabilization, as assessed by the strong decrease in the levels of epidermal growth factor receptor (eGFR), MeT and eRBB3 in Usp8-null mice27. The interaction between uSP8 and these receptors results in their deubiquitylation, thus rescuing them from

Box 1 | Kinomics and degradomics The task of characterizing the functions of all kinases and proteases and their bidirectional connections is daunting. Nevertheless, the recent availability of curated catalogues for both classes of enzymes has provided an excellent starting point for the global analysis of kinomes and degradomes3,5. Comparative bioinformatic analysis has also provided relevant information about evolutionary conservation, neofunctionalization and subfunctionalization events in both the kinase and protease fields. Innovative methods such as oligonucleotide or protein microarrays, activity‑based probes and quantum dot‑based biosensors, are now available for profiling kinases and proteases that are present in complex biological samples, including malignant tumours2,162. The strategies used to identify the substrates targeted by these enzymes can be classified into two general categories: proteomics‑based and genetics‑based approaches, with considerable overlap between them162,163. The first group includes methods involving phage displayed peptide libraries, peptide and protein array screens, yeast two‑hybrid‑based analyses or techniques such as isobaric tags for relative and absolute quantification (iTRAQ) and stable isotope labelling with amino acids in cell culture (SILAC) coupled to mass spectrometry. Genetics‑based approaches usually involve the generation of functional knockouts of kinases and proteases through gene targeting or RNA interference methods. Nevertheless, no single methodology will be sufficient to identify all in vivo substrates targeted by kinases and proteases. System‑wide approaches, termed kinomics and degradomics162,164, involving a combination of biochemical, proteomic, genetic, cell‑based and in vivo imaging studies, will be essential to identify these substrates and to better define the functional connections between these two fundamentally important and complex post‑translational regulatory systems. The resolution of three‑dimensional structures of kinases and proteases by using high‑throughput X‑ray crystallographic methods, homology modelling and ab initio predictions based only on protein sequence information will also facilitate the design of new inhibitors for targeting alone or in combination the specific kinases and proteases, the expression and function of which is altered in cancer.

degradation and allowing their recycling to the plasma membrane, although some studies have suggested that uSP8 might promote eGFR degradation28. The role of uSP8 in the control of cell proliferation is supported by the identification of an oncogenic fusion between uSP8 and the p85 subunit of PI3K in cells from a patient with leukaemia29. like uSP8, AMSH (also known as STAMBP) is an endosome-associated DuB that promotes eGFR recycling at the expense of lysosomal sorting 30. Interestingly, a recent RNA interference (RNAi) screen identified uSP18 as a new regulator of eGFR synthesis31. However, and unlike uSP8 and AMSH, uSP18 does not control eGFR levels by regulating the trafficking of ligand-activated internalized eGFR. Instead, uSP18 controls constitutive eGFR synthesis by increasing EGFR translation, which is a new protease-dependent mechanism of eGFR modulation31. Recent studies have unveiled additional interactions between kinases and DuBs (FIG. 3). uSP4 modulates the wnt pathway through its ability to interact with Nemolike kinase (NlK), negatively controlling this signalling pathway, which is commonly activated in cancer 32. uSP9X deubiquitylates the AMP-activated protein kinase (AMPK)-related kinases NuAK1 and MARK4, which are implicated in the regulation of cell polarity and proliferation, and modulates their phosphorylation and activation by lKB1 (REF. 33). Furthermore, uSP28 stabilizes components of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinase signalling, such as the checkpoint kinase CHK2, in response to DNA damage34. Moreover, uSP11 interacts with IKKα, an inhibitor of nuclear factor-κB (NF-κB), and modulates a p53 signalling pathway induced by tumour necrosis factor-α (TNFα)35, whereas uSP39 controls levels of Aurora B kinase and is essential for mitotic spindle checkpoint integrity36. last, the tumour suppressor cylindromatosis (CYlD) is another DuB protease that exhibits close links to kinases37. CYlD regulates cell proliferation, survival and migration by negatively modulating IKK–NF-κB and JuN N-terminal kinase (JNK) pathways37. CYlD also has a regulatory role in cell cycle progression, controlling entry into mitosis through direct interaction with Polo-like kinase 1 (REF. 38). This observation has suggested that CYlD may have dual functions in cancer, exhibiting anti-tumour properties but also pro-tumorigenic activities that are derived from its role as an enhancer of mitotic entry. Owing to central roles for several DuBs in the control of cell proliferation, their proteolytic activity must be tightly regulated. Several studies have revealed that kinases are directly involved in DuB control (FIG. 3). uSP7 — a key regulator of the p53–MDM2 pathway — is activated by phosphorylation of specific residues39, and uSP16 is phosphorylated at the onset of mitosis and regulates cell cycle progression by histone H2A deubiquitylation40. uSP44, another crucial regulator of mitosis, has been proposed to be activated through phosphorylation by mitotic CDKs or spindle checkpoint kinases41, whereas uSP15, uSP19, uSP28 and uSP34 are substrates of ATM and ATR and contribute to the regulation of diverse DNA damage checkpoints34,42,43.

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REVIEWS Proteases

Kinases Threonine

AGCs Atypical

Serine CAMKs

CK1s

CMGCs

Metallo Other

RGCs STEs

Cysteine

TKs Aspartic

TKLs

Figure 1 | Global representation of the crosstalk between the human kinome and the human degradome. Human | Cancer kinases and proteases are distributed into the families represented by the white and black sections Nature (detailsReviews can be found in Supplementary information S3 (figure)). Each individual enzyme is represented by a coloured line, and those with an extended length correspond to proteases (148; 26%) that are phosphorylated by kinases, or kinases (127; 24.5%) that are targeted by specific proteases. Classification of kinases and proteases is based on data from The Human Kinome website and The Mammalian Degradome Database (see Further information). AGCs, PKA, PKG and PKC kinases; CAMKs, calcium/calmodulin-dependent kinases; CK1s, casein kinase 1 group; CMGCs, CDK, MAPK, GSK3 and CLK kinases; RGCs, receptor guanylate cyclases; STEs, homologues of yeast Ste7, Ste11 and Ste20 kinases; TKs, tyrosine kinases; TKLs, tyrosine kinase-like enzymes.

Functional studies have shown that uSP28 is required to stabilize DNA damage pathway proteins such as CHK2, 53BP1 and claspin in response to genotoxic damage34. Conversely, A20 — a DuB protease frequently inactivated in B cell lymphomas — is phosphorylated by IKKβ, thus increasing its ability to inhibit the NF-κB signalling pathway 44. By contrast, other DuB family members such as CYlD are inhibited by phosphorylation, which leads to the temporary accumulation of ubiquitin chains on its target substrates45,46. Similarly, uSP8 activity is inhibited in a phosphorylation-dependent manner in interphase cells47, although other studies have shown that AKTmediated phosphorylation of this protease can result in its stabilization48. Notably, global phosphoproteome analyses have revealed that many DuBs, including 37 of 55 members of the uSP subfamily, are phosphorylated in vivo21,49–51. It is unclear what proportion of these sites

functionally regulates DuB activity, but these results indicate that the interplay between kinases and DuBs is very common and suggest that exploring these interactions will provide important information about the mechanisms controlling cell homeostasis. Separase. This cysteine protease triggers anaphase by the cleavage of sister chromatid cohesion in a DNAdependent manner52. To ensure faithful chromosome segregation and genome stability, separase activity is tightly regulated by the inhibitor securin53. Additionally, CDK1 and eRK2 efficiently phosphorylate and inhibit separase, and provide another level of regulation54. The functional relevance of interactions between separase and kinases has been reinforced after the finding of the mutual inhibition of separase and CDK1 by a two-step mechanism. First, separase is phosphorylated by CDK1 at a major



REVIEWS inhibitory site. Second, CDK1, through its regulatory cyclin B1 subunit, stably binds phosphorylated separase, leading to the formation of a complex that inhibits both protease and kinase11. Separase overexpression induces aneuploidy and mammary tumorigenesis in mice, and is a common event in many human cancers55,56.

Kinase–protease crosstalk in apoptosis The interplay between kinases and proteases also has a profound impact on apoptosis through the involvement of an array of proteolytic enzymes, such as caspases, calpains, granzymes and OMI proteases, which target kinases and are in turn regulated by phosphorylation14. Kinase–caspase crosstalk. Caspases can either activate or inactivate many different protein kinases (FIG. 4). Caspase-mediated cleavage of some kinases, such as ABl, triggers the nuclear translocation of a fragment that retains the intact catalytic domain and allows its recruitment to the apoptotic machinery 57. Caspases can also remove the auto-inhibitory domain of kinases and constitutively activate these enzymes12. This mechanism has been reported for many serine/threonine kinases, including RIPK1, Rho-associated protein kinase 1 (ROCK1), MST1 and several PKCs, as well as for some non-receptor TKs, including BMX, FYN and lYN12. The cleavage and activation of kinases by caspases can be blocked by inhibitors of caspase 3 and related proteases, which indicates that these enzymes are required for kinase activation12. Additionally, caspase 7 activates CHK1 whereas caspase 8 and caspase 10 are implicated in the activation of RIPK1, p21-activated kinase 2 (PAK2) and doublecortin-like kinase 1 (DClK1) 58,59. The mechanisms by which the caspase-activated kinases contribute to amplify the response to apoptotic stimuli are diverse and mainly depend on the characteristics of the downstream substrates (including the DNA repair machinery, apoptosis inhibitors and structural proteins) that are targeted by the activated kinases. The caspasemediated cleavage of some kinases is responsible for the morphological changes associated with apoptosis. For example, caspase-mediated activation of ROCK1 leads

Dependence receptors Structurally unrelated receptors that can induce cell death by apoptosis when unoccupied by ligand, and so create cellular dependence on their ligands. In the presence of ligand, these receptors mediate survival, differentiation and migration.

Apoptosome Multiprotein complex that mediates the activation of initiator caspases at the onset of apoptosis.

Catalytic activation

Removal of inhibitory domains

Catalytic inactivation

Complete degradation

Alterations in half-life

Protease

Kinase

Ectodomain shedding

Changes in cellular location

Changes in cellular location

Novel protein interactions

Novel biologically active fragments

Figure 2 | Functional interplay between kinases| and Nature Reviews Cancer proteases. Kinases switch protease activity on or off depending on phosphorylation status but can also modulate protease activity through changes in the indicated processes. Reciprocally, proteases switch kinases on or off through proteolytic processing reactions but can also influence kinase functions in more complex ways.

to changes in the actin cytoskeleton that contribute to the cell membrane blebbing characteristic of apoptotic cells60,61, whereas MST1 activation by caspase 3 facilitates its shuttling to the nuclear compartment where it phosphorylates histone H2B and induces chromatin condensation and DNA degradation62. Caspasemediated cleavage of kinases can also influence cell differentiation pathways. For example, activation of HPK1 by caspase 3 has a role in the differentiation of haematopoietic cells and the elimination of autoreactive T and B lymphocytes63,64. In contrast to caspase-mediated kinase activation, some caspase–kinase interactions can inactivate the targeted kinase (FIG. 4). For example, AKT and FAK are anti-apoptotic kinases that are cleaved and inactivated by caspases during apoptosis65,66. The successive cleavage of FAK by caspase 3 and caspase 6 contributes to morphological changes that are observed in cells undergoing apoptosis, as it interrupts the assembly of the focal adhesion complex. Similarly, calcium/calmodulin-dependent protein kinase IV (CaMKIV), BuB1 and BuBR1 are targeted and inactivated by caspase 3 and related proteases during apoptosis progression67,68, whereas ribosomal protein S6 kinase (p70S6K) is degraded in a caspase 8-dependent process69. Additionally, cleavage of ATM and DNA repair pathway kinase DNA-PKcs (also known as PRKDC) by caspases facilitates apoptosis by impairing DNA repair mechanisms in response to DNA damage70,71. Conversely, in response to apoptotic stimuli, eGFR is downregulated by caspases that cleave the receptor in its C-terminal region and abolish its ligand-induced pro-survival functions72. Interestingly, the caspase-mediated cleavage of other RTKs, such as ReT, MeT and eRBB2, which belong to the group of dependence receptors, proceeds in the absence of their respective ligands and results in the generation of proapoptotic receptor fragments73. Therefore, in addition to proteolytically inactivating these pro-survival RTKs, caspases convert them into generators of pro-apoptotic factors. Collectively, these results demonstrate that kinases are both substrates and effectors of caspases. Conversely, caspases are also targeted by kinases that switch on or off their pro-apoptotic functions (FIG. 4). The first demonstration that caspases are directly regulated by kinases was derived from the observation that the apoptotic initiator caspase 9 was inactivated by AKT-mediated phosphorylation74. Caspase 9 can also be phosphorylated at different inhibitory sites by other kinases, such as protein kinase A (PKA), CDK1, eRK1, eRK2, p38α, PKCζ, CK2 and DYRK1A, in response to various extracellular signals or cellular stresses75–77. The kinase-mediated inhibition of caspase 9 might involve an allosteric mechanism that affects its catalytic properties or alters its ability to interact with other proteins. Consistently, phosphorylation of caspase 9 by CK2 protects the protease from cleavage and activation by other caspases78, whereas PKA blocks its recruitment to the apoptosome79. This negative regulation of caspase 9 is important in cancer as it protects mitotic cells against apoptosis and promotes cell survival during tumorigenesis80. Caspase 8 can also be inactivated by kinases, including SRC10.

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REVIEWS TNFα

EGFR

TRAF complex

USP18 Translation

Recycling

RIPK1

MKK7

Ub Lysosome

AMSH

USP8

P

CYLD

USP8 CDKs USP8

Ub

NUAK1

MARK4

P

JNK

TAK1

JUN

NLK

Ub

β-catenin

LKB1 P

IKKα

IKKβ P P

MARK4

USP4

Ub RIPK1

K48 A20

P

A20

P NF-κB IκBα NF-κB

BCL-3

IκBα

P

BCL-3

Ub

IκBα USP15

NLK MARK4

NUAK1

K63

Ub

Ub

USP9X NUAK1

NEMO

CYLD

AKT

Proteasome

Ub RIPK1

BCL-3 NF-κB

USP15 P USP19 P USP34 P ATM and ATR

P

P

TNFα

Ub

TRAF complex IKKα

P

USP28

p53

CHK2 P

USP11

P

IKKα USP7

p53

β-catenin

JUN target genes

USP16 CDKs P

H2A

USP16

Wnt target genes mRNA Splicing

H2A Ub

NF-κB target genes

USP39

DNA damage

P

USP7 p53

Ub APC Cdc20

Aurora B

MDM2

APC Cdc20 Mad2

P

Ub

USP44 CDKs? USP44

Wnt

Figure 3 | Overview of the crosstalk between kinases and deubiquitylases (DUBs). Examples of DUBs (dark blue Reviews boxes) that target kinases (pink boxes) and cause their activation or inactivation; DUBs are switchedNature on (yellow P) or| Cancer off (orange P) by kinase-dependent phosphorylation. Ub in green corresponds to lysine 48 (K48)-linked chains targeting proteins to the proteasome, whereas blue Ub indicates non-K48-linked chains. DUB–kinase crosstalk can affect kinase levels (top left). Ubiquitin-specific protease 8 (USP8) (stabilized by AKT and inhibited by mitotic kinases), USP18 and AMSH stabilize epidermal growth factor receptor (EGFR), promoting its recycling or regulating its translation, and USP9X targets NUAK1 and MARK4 and modulates their activation by LKB1. DUB–kinase interplay also affects gene transcription pathways (top middle). CYLD is inhibited by phosphorylation and negatively modulates IKK–nuclear factor-κB (NF-κB) and JUN N-terminal kinase (JNK) pathways by targeting transforming growth factor-β-activated kinase 1 (TAK1) and MKK7; USP15 stabilizes inhibitor of NF-κB (IκBα) to regulate the NF-κB pathway; A20, which possesses K63-specific DUB activity, is activated by IKKβ-mediated phosphorylation and negatively regulates the NF-κB pathway by editing the Ub chain of RIPK1; USP4 modulates the Wnt pathway through interaction with Nemo-like kinase (NLK). DUB–kinase crosstalk also affects the DNA damage response (right middle). USP11 stabilizes IKKα in response to tumour necrosis factor-α (TNFα); nuclear USP28 stabilizes CHK2 in response to DNA damage; USP15, USP19, USP28 and USP34 are phosphorylated in response to genotoxic stress and modulate DNA damage checkpoints; USP7 is also phosphorylated and regulates the p53–MDM2 pathway. DUB–kinase interplay also has a role in cell cycle control (bottom). USP16 and USP44 can be activated by phosphorylation in the nucleus and regulate cell cycle progression, whereas USP39 controls Aurora B levels. APC, adenomatous polyposis coli; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; CDKs, cyclin-dependent kinases; H2A, histone 2A; TRAF, TNF receptor-associated factor.

Interestingly, SRC phosphorylation of caspase 8 also facilitates its interaction with pro-migratory proteins containing phosphotyrosine binding domains, such as the p85 subunit of PI3K, which allows this pro-apoptotic protease to become a cell motility factor 10,81. Similarly, lYN phosphorylates and inactivates caspase 8, and contributes to the inhibition of apoptosis of inflammatory

cells82. p38 MAPK also switches off caspase 8 and other caspases in inflammatory cells by direct phosphorylation, impairing their capacity to induce apoptosis83. In contrast to these examples of kinase-mediated inhibition of caspases, phosphorylation of these proteases can also induce their activation (FIG. 4). This is the case for caspase 1, which is activated by RIPK2 (REF. 84),



REVIEWS Actin bundle

Membrane blebbing

P P

Actin contraction

P ROCK1

ROCK2

ROCK1

ROCK2

EGFR

ERBB2

ERBB2

P

FAK

BH3-only

BCL-2 or BCL-XL MST1

Caspase 3

CaMKII

CK2

Caspase 2

Granzyme B

Caspases 2, 3, 8 and 9

Caspase 8

p70 S6K

ATM Caspases 2, 3, 6, 7, 8 and 10

AKT

Caspase 3

PKCδ

p53

PKCδ

Translation SRC Death receptors

p38

PKCδ

Caspase 3

BH3-only Perforin

Granzyme B Granzyme B pathway

Intrinsic pathway

Histone H2B

Caspase 2

Chromatin stability

P

Lamin B Nuclear envelope integrity

Nuclear fragmentation

Caspase 9

Extrinsic pathway BCL-2 or BCL-XL

DNA-PKcs

Caspase 2

LYN

Caspase 8

MST1

DNA repair

ERK

BAX or BAK

CDK1 PKA

PKCζ Caspase 9

Cytochrome c release

DYRK1A

Apoptosome

AKT p38

Granzyme B

Caspase 9

CK2

Caspase 9

Figure 4 | Interplay between kinases and apoptotic proteases. Interactions of caspases and granzyme with kinases Nature B Reviews | Cancer during apoptosis involve kinase cleavage and protease phosphorylation. Active caspases are shown in dark blue boxes and inactive caspases in purple boxes. Additionally, apoptotic proteases convert inactive kinases (yellow boxes) into active forms (pink boxes). Kinase–protease crosstalk can activate apoptosis (bottom left). Apoptosis induction by death receptors or by BH3-only proteins binding to BCL-2 or BCL-XL results in cytochrome c release from mitochondria and apoptosome formation. Both the apoptosome and granzyme B activate caspase 9, which in turn activates downstream effector caspases that ultimately induce apoptosis. Caspase 8 and caspase 9 are inactivated by the indicated kinases. Apoptotic morphological changes are induced by kinase–protease crosstalk (top left). Caspase 3 and granzyme B remove the auto-inhibitory domain of Rho-associated protein kinase 1 (ROCK1) and ROCK2, the activation of which contributes to membrane blebbing through phosphorylation of actin bundles. Cell surface and signal transduction events are also affected by kinase–protease interplay in apoptosis (top middle). Caspases and granzyme B proteolytically inactivate kinases such as AKT, focal adhesion kinase (FAK), ribosomal protein S6 kinase (p70S6K) and epidermal growth factor receptor (EGFR). In some cases, the caspase-mediated cleavage of receptor tyrosine kinases (RTKs), such as ERBB2, results in the generation of proapoptotic BH3-only fragments. Caspase 2 is inhibited by casein kinase 2 (CK2) and calcium/calmodulin-dependent protein kinase II (CaMKII). Kinase–protease crosstalk can also induce apoptotic nuclear responses (top right). Caspases generate active forms of MST1 or protein kinase Cδ (PKCδ), which translocate to the nucleus and contribute to chromatin condensation or nuclear and DNA fragmentation. Additionally, ataxia telangiectasia mutated (ATM)-inactivating cleavage by caspases facilitates apoptosis by impairing DNA repair mechanisms. Caspase 3 is activated by PKCδ, whereas caspase 2 is activated in the nucleus by DNA-PKcs (also known as PRKDC) following DNA damage. CDK1, cyclin-dependent kinase 1; PKA, protein kinase A.

caspase 3 (the proteolytic activity of which is enhanced by PKCδ-mediated phosphorylation85), caspase 9 (the activation of which is regulated by ABl in response to genotoxic stress86) and caspase 2 (which is activated in the nucleus by phosphorylation after DNA damage87). Caspase 2 phosphorylation is carried out by DNAPKcs in a large nuclear protein complex consisting of DNA-PKcs, PIDD and caspase 2, which is called the DNA-PKcs-PIDDosome. This phosphorylation event and the catalytic activity of caspase 2 help to maintain a G2/M DNA damage checkpoint and DNA repair 87. The DNA-PKcs-PIDDosome is a new kinase–protease protein complex that affects the cellular response to

DNA damage, which is of great relevance in cancer. It is also noteworthy that phosphoproteome analyses have revealed that other caspases, such as caspase 4, caspase 6, caspase 7 and caspase 10, are phosphorylated in vivo50,51, although the functional consequences of these newly identified interactions between caspases and kinases remain to be explored. Other apoptotic protease–kinase interactions. During apoptosis, several hundred proteins, including kinases, undergo controlled proteolysis to minimize damage to neighbouring cells and avoid unwanted immune responses. Caspases carry out many of these proteolytic

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REVIEWS events that weaken key cellular structures and lead to the destruction of the apoptotic cell14. However, other proteases contribute to apoptosis and are also involved in mutual interaction with kinases. For example, CaMKIV is cleaved and inactivated by calpain 1 in neuroblastoma cells undergoing apoptosis68, and calpain 2 proteolytically inactivates FAK during SRC-induced transformation, resulting in the loss of focal adhesion structures88. In addition to apoptotic cysteine proteases, such as caspases and calpains, serine proteases, including granzyme B and OMI, function in concert with kinases during programmed cell death. Granzyme B causes the apoptosis of virally infected or tumour cell targets by directly cleaving and activating ROCK2, which in turn contributes to membrane blebbing in a caspase-independent manner 13. Granzyme B also targets DNA-PKcs, generating unique fragments that can induce apoptosis89, and cleaves TKs such as fibroblast growth factor receptor 1 (FGFR1) and ABl, inactivating pro-growth signals and causing cell death90. The anti-apoptotic FAK is also inactivated by cytotoxic T lymphocyte-derived granzyme B during apoptosis91. In addition, OMI is a pro-apoptotic mitochondrial protease, the activity of which is negatively modulated by AKT-mediated phosphorylation92. However, it is activated by kinases such as lATS1 (also known as wARTS), which in turn are substrates for this protease8. Notably, in addition to these intracellular events derived from apoptotic protease–kinase interactions, several metalloproteinases, including ADAMs and matrix metalloproteinases (MMPs), modulate extracellular events by shedding cell surface RTKs in response to apoptotic stimuli6.

Kinase–protease crosstalk in metastasis The direct and mutual interactions between caspases or calpains and different kinases have an important impact on the regulation of cell migration and invasion that is linked to cancer progression93,94. likewise, ADAMs and presenilins are engaged in shedding and RIPping processes in which these proteases sequentially act on many RTKs, modifying their signalling properties and facilitating tumour invasion6,15,16. ADAMs are transmembrane proteins that are phosphorylated in their cytoplasmic tails, but function by cleaving their substrates at or in the outer layer of the cell membrane6. Additionally, proteases from other families, including furin and MMPs, establish bidirectional interactions with kinases that contribute to the metastatic properties of cancer cells95.

Furin Member of a group of propotein convertases that catalyse the maturation of precursors for many secreted and transmembrane proteins by cleavage at Arg-X-Lys/ Arg-Arg sites.

Caspases, calpains and kinases in cell migration. The relevance of caspase–kinase interactions in cell migration was first illustrated by the finding that caspase 8 promotes motility in a manner dependent on phosphorylation by SRC kinases and its subsequent association with pro-migratory proteins 81. Further analysis has shown that integrin-mediated adhesion promotes caspase 8 phosphorylation and its subsequent engagement in invasive processes93,96. Caspase 8 also interacts with non-receptor TKs, such as SRC and FAK, enhancing eGF signalling and cleavage of FAK substrates, thereby promoting cell migration and metastasis93,97.

The proteolytic activity of calpains on certain kinases also contributes to remodelling of the actin cytoskeleton and cell migration 7,98. Calpain 2 proteolytically inactivates FAK during SRC-regulated transformation, resulting in the disassembly of focal adhesion structures88. Notably, FAK can also function as an upstream mediator of calpain activity, and so contribute to the promotion of tumour invasion99. Additionally, and similar to caspases, calpains remove inhibitory domains and generate constitutively active forms of kinases such as PKC, GSK3 and CaMKII, which are involved in signal transduction pathways that are relevant for migration and invasion100–102. Also, as is the case for caspases, phosphorylation at several sites controls the activities of calpains7. Calpain 2 activation by eRK is required during processes such as focal adhesion complex turnover and cell migration103. Similarly, PKCι promotes migration and invasion of lung cancer cells through the phosphorylation of calpains104. By contrast, PKA inhibits calpain 2 by phosphorylation at Ser369. This residue is located in the interface region between calpain domains III and IV, and when phosphorylated it enables new interactions that restrict the movement of both domains and maintains the protease in an inactive state105. The crosstalk between kinases and calpains could have important consequences in cancer pathogenesis, as calpain overexpression is associated with tumour invasion and metastasis106,107. Furin, kinases and metastasis. Several studies have shown the close bidirectional links between furin and kinases. Furin phosphorylation is crucial for its proper intracellular location and recycling 108. Reciprocally, several kinases are activated by the proprotein convertase activity of furin in a process that is important for different stages of cancer, including metastasis109. Furin is a major insulin-like growth factor 1 receptor (IGF1R) convertase, as demonstrated through the overexpression of α1-antitrypsin Portland (α1-PDX), a bioengineered furin inhibitor, and the knock down of furin in colon cancer cells109. Colorectal tumour cells in which furin-mediated processing of IGF1R is abolished by α1-PDX overexpression have a reduced ability to form liver metastases in mice109, indicating the importance of furin–kinase crosstalk and suggesting that disruption of this communication could be a new strategy for preventing colorectal cancer liver metastasis. Proteases and kinases in shedding and RIPping. Surface shedding affects many proteins, including most RTK ligands, but it can also directly regulate the activity of RTKs6,15,72. ectodomain processing can make these receptors unavailable for ligand binding or generate decoys that sequester soluble ligands. Alternatively, shedding may constitutively activate ligand-independent receptors110, or modify their properties, if the initial proteolytic step is followed by a RIPping reaction that is mediated by the γ-secretase protease complex 111. This second proteolytic cleavage in the transmembrane region releases the cytoplasmic domain of the receptor and in some cases allows it



REVIEWS to enter the nucleus and participate in the transcriptional regulation of target genes. An excellent example is eRBB4, which undergoes shedding and RIPping to generate the B4 intracellular domain (B4ICD) fragment, which translocates into the nucleus, where it functions either as a chaperone or as a cofactor for different transcription factors112. The B4ICD fragment also exhibits pro-apoptotic activity in breast cancer cells through an association with the mitochondria through its BH3 domain113. RIPping is also required for nuclear signalling through other RTKs, such as IGF1R, which on cleavage by the γ-secretase complex releases a C-terminal fragment with a direct proapoptotic role or with dominant-negative functions that abolish the survival signal that is mediated by the receptor 114. Similarly, insulin receptor processing by γ-secretase generates a soluble intracellular domain that localizes to the nucleus and potentially modulates insulin-induced signalling events115. ADAM17 is the principal enzyme responsible for RTK ectodomain shedding and has been reported to cleave neurotrophic tyrosine kinase receptor 1 (NTRK1), vascular endothelial growth factor receptor 2 (VeGFR2), eRBB4, MeT, KIT and colony stimulating factor 1 receptor (CSF1R), although other ADAMs such as ADAM9, ADAM10 and ADAM15 have sheddase activity for RTKs, including FGFR2, eRBB2, AXl and eph receptor B2 (ePHB2) (reviewed in REFS 6,72 ). likewise, proteases of different families can also shed RTKs, such as FGFR1 (which is targeted by MMP2), ePHB2 (which is targeted by MMP2 and MMP9), AXl (which is targeted by MT1-MMP (also known as MMP14)), VeGFR2 (which is targeted by matriptase) and eGFR (which is targeted by a matriptase–prostasin serine protease cascade)116–120. The processes of shedding and RIPping are highly regulated at different levels, including kinase-mediated events that can modulate protease function directly or facilitate their interaction with other proteins. Therefore, in an interesting example of kinase–protease reciprocal interactions, some ADAMs bind and activate kinases that in turn phosphorylate and activate ADAMs involved in shedding. This is the case for ADAM9, which associates with the catalytic domains of PKCδ and SRC, and on phosphorylation, is activated for shedding processes 121. ADAM10 also interacts with several kinases, but its involvement in a complex mechanism for displacement of the membrane-proximal ePHA3 RTK kinase domain is particularly interesting 122. This promotes the correct orientation of ADAM10 so it can subsequently shed the ephrin-A ligand (bound to ePHA3) from the presenting cell, ensuring that only ligands bound to the RTK are recognized and cleaved by ADAM10. Additionally, ADAM12 binds and activates SRC and other kinases, such as YeS1, PI3K and PKCδ, which in turn phosphorylate ADAM12 and induce a conformational change that causes its translocation to the cell surface 123. ADAM15 also associates with different tyrosine kinases (such as SRC, lCK, HCK, FYN and ABl) and its phosphorylation influences its function124. For example, the shedding

activities of ADAM15B, a splice variant of ADAM15, are enhanced by SRC binding and phosphorylation125, whereas the activation of ADAM17 is dependent on eRK-mediated phosphorylation126. The activity of other proteases that are involved in shedding and RIPping processes is modulated by kinases. MMP2 is inhibited by PKC-dependent phosphorylation127, whereas MT1-MMP is phosphorylated at its cytoplasmic tail by SRC kinases, facilitating tumour cell migration128,129. Presenilin 1 (PS1) and PS2 are also regulated by phosphorylation through complex mechanisms. Phosphorylation of PS1 by different kinases elicits changes in protease stability, cellular localization and function, affecting the interaction with β-catenin and the cleavage of substrates such as N-cadherin130–132. Additionally, PS1 and PS2 phosphorylation inhibits their cleavage by caspases and retards progression of apoptosis133,134. The functional effect on cancer of these protease– kinase interactions that occur during shedding and RIPping processes are diverse and dependent on the function of the shed protein. It is well established that shedding activities that solubilize RTKs regulate multiple activities in the tumour microenvironment, including several that are especially relevant for invasion and metastasis. For example, MeT — a widely studied RTK owing to its links with tumour invasion — is a target of a series of sequential presenilindependent proteolytic cleavages that downregulate the receptor independently of ligand stimulation and result in the inhibition of MeT-dependent invasive growth of transformed cells135. Interestingly, forced induction of presenilin-dependent MeT proteolysis by using MeT inhibitory antibodies results in the inhibition of MeT-dependent invasive growth and could be of therapeutic interest in MeT-driven malignancies135. Additionally, VeGFR2 is shed from the cell surface by ADAM17, decreasing its availability on the cell surface and generating soluble decoys that regulate angiogenesis and impair the generation of metastasis136. These are illustrative examples of protease-mediated events acting on kinases that have a negative impact on invasion and metastasis. Conversely, the relevance of shedding events for facilitating metastasis is supported by the finding that two distinct metalloproteinases, MMP1 and ADAMTS1, proteolytically engage eGF-like ligands and orchestrate an eGFRdependent paracrine signalling cascade that favours the development of bone metastasis95. These findings support the idea that combined inhibition of metalloproteinases and RTK signalling may be effective for cancer therapy.

Clinical implications of kinase–protease crosstalk Kinases and proteases are attractive targets for therapeutic intervention in cancer. Considerable success has already been achieved with monoclonal antibodies and small molecule inhibitors that target diff erent kinases and are currently used for treating different cancers137,138. Progress in the protease field has not been as rapid as that in the kinase field, but

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REVIEWS Trastuzumab Humanized monoclonal antibody that interferes with ERBB2 signalling and is currently used for the treatment of breast cancer.

recent successes with proteasome inhibitors for the treatment of haematological malignancies139 has stimulated new efforts aimed at inhibiting other proteases that are dysregulated in cancer 140–143. Nevertheless, looking beyond the studies singularly focused on either kinases or proteases, the catalogue of mutual interactions between both types of enzymes also has important clinical consequences (TABLE 1). There are specific examples in which the recognition of kinase–protease crosstalk has already provided new opportunities for cancer treatment. This is the case of the interplay between ADAMs and the eGFR family. Interestingly, analysis of serum from cancer patients has revealed the presence of proteolysed soluble forms of the eGFR family member eRBB2 (REF. 110) . The presence of soluble or truncated forms of eRBB2 is linked to poor prognosis and reduced effectiveness of therapies against eRBB2, as these may neutralize the currently used eRBB2specific antibodies 144. These findings suggest that the addition of protease inhibitors to RTK inhibitors could result in synergistic anti-tumour activity 145. The recent confirmation of a synergistic inhibition of breast cancer cell growth with a dual eGFR and eRBB2 inhibitor (Gw2974) and an ADAM10 and ADAM17 inhibitor (INCB7839) 146, together with similar findings using selective eGFR and ADAM17 inhibitors in colorectal cancer cells147, has provided a new approach to improve the currently available RTKdirected therapies. An ongoing clinical trial is already exploring the value of INCB7839-mediated ADAM inhibition in combination with trastuzumab (eRBB2specific) for the treatment of metastatic breast cancer

(NCT00864175; The Incyte website, see Further information). In addition, the identification of functional links between ADAM15B and SRC kinases has suggested that inhibitors of the interaction between these proteins could represent a new therapeutic option to treat patients with breast cancer with dysregulated ADAM15 (REF. 125) . Furthermore, the observation that ADAM9 promotes neovascularization through the shedding of RTKs suggests that ADAM9 and RTK inhibitors could be combined to interfere with angiogenesis148. likewise, the close cooperation between SRC-mediated phosphorylation of MT1-MMP and eGFR-dependent tumour cell proliferation and invasion suggests that targeting this kinase-dependent event could be an innovative therapeutic strategy 128. Similarly, targeting MMP1 and ADAMTS1 metalloproteinases combined with the inhibition of eGFR signalling in the bone stroma has been proposed as a new approach for reducing the risk of bone metastasis in patients with breast cancer 95. Besides these examples involving metalloproteinases and tyrosine kinases, there are additional situations in which the targeting of different protease– kinase interactions might offer opportunities for therapeutic intervention in cancer. The DuB family is especially relevant in this regard. The observation that overexpression of the serine/threonine kinase NlK promotes nuclear accumulation of uSP4, which is a negative regulator of cell proliferation, has suggested a new approach for blocking the wnt signalling pathway that is commonly dysregulated in many malignancies 32. Additionally, the fact that the reduction of uSP8 and AMSH levels enhances internalization

Table 1 | Possibilities of combined anticancer therapies derived from kinase–protease crosstalk* Targeted kinase

Targeted protease

Tumour type

refs

EGFR and ERBB2

ADAM10 and ADAM17

Breast cancer

146

EGFR

ADAM17

Colorectal cancer

147

EGFR

ADAM10 and ADAM17

Non-small-cell lung cancer

145

SRC

ADAM15

Breast cancer

125

SRC

MT1-MMP

Fibrosarcoma cells

128

EGFR

MMP1 and ADAMTS1

Bone metastasis

EGFR

USP8 and AMSH

EGFR-dependent tumours

EGFR

USP18

EGFR-dependent tumours

EGFR

Proteasome

Several cancer cell lines

AKT

Proteasome

Prostate cancer

153

AKT

Proteasome

Hepatocellular carcinoma

154

JNK

Proteasome

Pancreatic cancer

155

PI3K–AKT pathway

Proteasome

Pancreatic cancer

155

p38α MAPK

Proteasome

Multiple myeloma

156

ERBB2

γ-secretase

Breast cancer

157

γ-secretase

T cell acute lymphoblastic leukaemia

158

PI3K–AKT pathway

95 149 31 150–152

ADAM, a disintegrin and metalloproteinase; EGFR, epidermal growth factor receptor; JNK, JUN N-terminal kinase; MMP, matrix metalloproteinase; USP, ubiquitin-specific protease. *These therapeutic approaches have only been validated in in vitro or in vivo models of cancer, with the exception of the combined targeting of ERBB2, ADAM10 and ADAM17, which is now in a clinical trial in patients with breast cancer.



REVIEWS and degradation of several RTKs, together with the observation that both proteases are upregulated in several tumour types, suggests that these DuBs might be druggable targets in cancer 149. likewise, as uSP18 depletion downregulates eGFR, inhibition of this cysteine protease might be a useful treatment for eGFR-dependent tumours 31. Other DuBs, such as uSP7, uSP28 and CYlD, which exhibit close links with kinases and the expression or activity of which is dysregulated in cancer, are attractive pharmacological targets in combination with kinase inhibitors149. Nevertheless, the finding that these three proteases may function as either oncoproteins or tumour suppressors depending on the genetic or cellular context, provides additional complexity to targeting them in cancer. The many interactions between the proteasome and kinases have also stimulated the search for new combined cancer therapies. Therefore, the combination of bortezomib-mediated proteasome inhibition and eGFR inhibition with either small molecules or antibodies has a synergistic anti-proliferative and pro-apoptotic activity in different human cancer cells150–152. Furthermore, the combined treatment of prostate cancer cells with bortezomib and the AKT inhibitor perifosine is more effective than treatment with either compound alone 153. Additional studies have shown that targeting the AKT signalling pathway overcomes drug resistance to bortezomib in hepatocellular carcinoma cells 154. Similarly, inhibition of the JNK and PI3K–AKT pathways increases the anti-tumour effects of proteasome blockade in pancreatic cancer cells155, whereas inhibition of p38α MAPK enhances the bortezomib-induced cytotoxicity against myeloma cells and the inhibition of tumour growth in mice156. Collectively, these preclinical studies provide a conceptual framework for clinical trials aimed at increasing sensitivity or overcoming resistance to compounds the efficacy of which is limited as single therapeutic agents. The combination of kinase and γ-secretase inhibition may also be effective in some tumour types. The observation that eRBB2 blockade with trastuzumab sensitizes breast cancer cells to a γ-secretase inhibitor has led to the proposal of therapies combining trastuzumab with compounds targeting the γ-secretase complex in patients with eRBB2-positive breast cancer 157. In addition, inhibitors of the PI3K–AKT pathway in combination with γ-secretase inhibitors could be useful for the treatment of a subset of patients with T cell acute lymphoblastic leukaemia 158. The disruption of furin–kinase communication also offers new therapeutic opportunities in cancer, as selective blockade of furin inhibits processing of IGF1R and reduces the metastatic potential of human colorectal tumour cells 109. Similarly, caspase–kinase crosstalk might have important clinical implications in cancer treatment. For example, strategies aimed at increasing the activity of pro-apoptotic caspases by using antagonists of their endogenous inhibitors in combination with eRBB2 inhibitors have proved to be effective in

breast cancer cells and could be of interest in patients with eRBB2-positive breast cancer 159. likewise, the concomitant targeting of RTKs and caspase inhibitors of the inhibitor of apoptosis (IAP) family overcomes resistance to RTK inhibitors in human glioblastoma cells, suggesting that blockade of IAPs may improve the clinical outcome of patients with glioblastoma multiforme treated with RTK inhibitors160. However, the recent discovery of paradoxical pro-metastatic functions of caspase 8 derived from its interaction with non-receptor TKs implies that clinical strategies based on increasing caspase levels in cancer patients would not be effective in all cases and might even lead to tumour progression and dissemination93. This example illustrates that the complexity of caspase– kinase interactions — involving multiple events of caspase-mediated activation or inactivation of kinases as well as kinase-mediated activation or inactivation of caspases — makes a careful analysis of the putative benefits derived from their combined targeting necessary.

Conclusions and perspectives Our view of the crosstalk between kinases and proteases has come a long way since Fischer and Krebs fortuitously discovered in 1955 that calcium was necessary for the activation of glycogen phosphorylase kinase through proteolytic processing that is mediated by a calcium-dependent protease161. In the ensuing years, studies of either kinases or proteases have converged in the recognition of the multiple links between both posttranslational regulatory systems. This first attempt to examine in-depth the kinase–protease interactions and to define their functional effect in cancer has revealed the enormous diversity and complexity of their bidirectional communication tactics. Many kinases undergo proteolytic processing reactions that result in the activation or inactivation of their signalling properties. Conversely, several intracellular proteases and some transmembrane enzymes with cytoplasmic tails are switched on or off by kinases, and contribute to the processing of cellular signals. Moreover, this crosstalk extends beyond direct kinase–protease interactions, and is influenced by their corresponding endogenous regulators, including phosphatases (BOX 2) and protease inhibitors. The evidence discussed above includes examples of kinases and proteases working together for the acquisition of uncontrolled proliferation, resistance to apoptosis, and migratory and invasive phenotypes that are characteristic features of malignant cells. Nevertheless, the evidence for the assignation of roles for kinase and protease-mediated events in cancer is still incomplete, and is restricted to specific situations, or can even be contradictory, depending on tissue type and tumour microenvironment. Therefore, further studies will be necessary for their functional and clinical validation. Many challenges must also be addressed before this basic information can be translated into the clinic. we have discussed some cases in which the lack of success of clinical trials using

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REVIEWS Box 2 | Phosphatase–protease crosstalk in cancer Protein phosphatases target proteases, turning their proteolytic activity on or off. Caspase 3, caspase 8 and caspase 9 are activated through dephosphorylation by PP2A, SHP1 (also known as protein‑tyrosine phosphatase, non‑receptor type 6 (PTPN6)) and PP1α (also known as PPP1CA), respectively, which facilitates apoptosis82,165,166. By contrast, calpain 1 and calpain 2 are inactivated by phosphatases94. Reciprocally, phosphatases are switched on or off by proteases. Phosphatases such as PP2A, PTP‑PEST (also known as PTPN12) and CDC25A are activated by caspase‑mediated cleavage167, whereas caspase 3 inactivates the PTEN phosphatase168. Likewise, calpains can inactivate PTPs through proteolytic degradation, although in some cases calpains increase PTP activity169. Like receptor tyrosine kinases, PTP extracellular domains can undergo shedding mediated by a disintegrin and metalloproteinase (ADAM) family members, and some PTPs are further processed by complex proteolytic events involving γ‑secretase, which releases a catalytic fragment that affects transcription in the nucleus170,171. Phosphatase and protease interplay also affects the different stages of cancer. Direct interactions between phosphatases and proteases may influence tumour cell proliferation. For example, interactions between separase and PP2A downregulate PP2A, promoting mitotic exit172. Likewise, several phosphatases are degraded by the proteasome, thereby modulating their functions during cell cycle progression. In this regard, it is interesting that PTEN subcellular localization is regulated by ubiquitin‑specific protease 7 (USP7)‑mediated deubiquitylation through a network that is disturbed in cancer173. Overexpression of USP7 causes nuclear exclusion of PTEN, which is associated with more aggressive prostate cancer and leukaemia173. The bidirectional interactions between caspases and phosphatases during apoptosis are also altered during tumour progression12. Phosphatase and protease crosstalk also occurs during tumour cell migration and invasion, especially through calpains and proteases that activate shedding and regulated intramembrane proteolysis (RIPping) events94,171. Understanding the mechanisms underlying phosphatase–protease crosstalk has provided new therapeutic opportunities for cancer. Therefore, the observation that PP2A suppresses cell invasion through calpain dephosphorylation suggests that PP2A is an attractive target to block calpain‑dependent cancer cell migration94. Likewise, blockade of PTPμ fragments that are generated by proteolysis decreases glioblastoma cell migration and could be of therapeutic value in glioblastoma multiforme171.

a single targeted agent for treating cancer patients has probably resulted from the lack of recognition of the kinase–protease crosstalk in these malignancies. Accordingly, it will be necessary to precisely identify the protein kinases that are involved in the regulation of protease activity and the proteases involved in the proteolytic processing of kinases in each specific condition. This may be especially important in the case of complex families of proteases, such as DuBs, that contain many members that are phosphorylated in vivo and commonly dysregulated in malignant tumours, but for which the functional relevance of

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Acknowledgements

We thank all members of our laboratories for their helpful comments on the manuscript and apologize for the omission of relevant works owing to space constraints. C.L-O. is supported by grants from Ministerio de Ciencia e InnovaciónSpain, European Union-Microenvimet and Fundación M. Botín. T.H. is supported by grants from the National Cancer Institute and is a Frank and Else Schilling American Cancer Society Research Professor. The Instituto Universitario de Oncología is supported by Obra Social Cajastur and Acción Transversal del Cáncer RTICC.

Competing interests statement

The authors declare no competing financial interests.

DATABASES ClinicalTrials.gov: http://clinicaltrials.gov/ NCT00864175 National Cancer Institute Drug Dictionary: http://www.cancer.gov/drugdictionary/ bortezomib | perifosine UniProtKB: http://www.uniprot.org ABL | ADAM9 | ADAM10 | ADAM15 | ADAM17 | AMSH | ATM | ATR | calpain 2 | caspase 1 | caspase 2 | caspase 3 | caspase 6 | caspase 7 | caspase 8 | caspase 9 | caspase 10 | CDK1 | CHK2 | CSN5 | CYLD | DNA-PKcs | EGFR | ERBB2 | FAK | furin | granzyme B | IGF1R | JAK2 | MARK4 | MET | MMP2 | MST1 | MT1-MMP | NLK | NUAK1 | OMI | p27 | PRKD1 | PS1 | PS2 | ROCK1 | separase | SRC | TNFα | USP4 | USP7 | USP8 | USP9X | USP11 | USP16 | USP18 | USP28 | USP39 | USP44 | VEGFR2

FURTHER INFORMATION Carlos López-Otín’s homepage: http://degradome.uniovi.es/lab.html Incyte: http://www.incyte.com/ The Human Kinome: http://kinase.com/human/kinome/ The Mammalian Degradome Database: http://degradome.uniovi.es/

SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (table) | S3 (figure) all lInks are aCTIve In The OnlIne pDF
