Syntenin mediates SRC function in exosomal cell-to-cell communication.

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.

Roles of exosomes in metastatic colorectal cancer.

Metastases remain a major cause of cancer morbidity and mortality. This is a multistep process that involves aberrant cell communication, leading to tumor cell dissemination from the primary tumor and colonization of distinct organs for secondary tumor formation. The mechanisms promoting this pathological process are not fully understood, although they may be of obvious therapeutic interest. Exosomes are small cell-secreted vesicles that contain a large variety of proteins, lipids, and nucleic acids with important signaling activities, and that represent an evolutionarily conserved mechanism for cell-to-cell communication. Not surprisingly, exosome activities have gained strong interest in cancer biology and might play essential roles in metastasis development. Here, we will describe recent findings on the role of exosomes in cancer metastasis formation, particularly in colorectal cancer (CRC). We will also discuss the potential therapeutic value of these vesicles in metastatic cancer.

CBL controls a tyrosine kinase network involving AXL, SYK and LYN in nilotinib-resistant chronic myeloid leukaemia.

A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.

Contribution of phosphoproteomics in understanding SRC signaling in normal and tumor cells.

The membrane-anchored, non-receptor tyrosine kinase (non-RTK) SRC is a critical regulator of signal transduction induced by a large variety of cell-surface receptors, including RTKs that bind to growth factors to control cell growth and migration. When deregulated, SRC shows strong oncogenic activity, probably because of its capacity to promote RTK-mediated downstream signaling even in the absence of extracellular stimuli. Accordingly, SRC is frequently deregulated in human cancer and is thought to play important roles during tumorigenesis. However, our knowledge on the molecular mechanism by which SRC controls signaling is incomplete due to the limited number of key substrates identified so far. Here, we review how phosphoproteomic methods have changed our understanding of the mechanisms underlying SRC signaling in normal and tumor cells and discuss how these novel findings can be used to improve therapeutic strategies aimed at targeting SRC signaling in human cancer.

Analysis of SRC oncogenic signaling in colorectal cancer by stable isotope labeling with heavy amino acids in mouse xenografts.

The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [(13)C(6)]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.

[Analysis of oncogenic signaling induced by tyrosine kinases in tumors by SILAC-based quantitative proteomic approach]. / Analyse in vivo de la signalisation tumorale induite par les tyrosine-kinases par protéomique quantitative.

Protein tyrosine kinases (TK) transmit intracellular signaling induced by many extracellular stimuli resulting in cell growth or adhesion. Deregulation of their activity leads to malignant cell transformation that plays an important role in human cancer. The signaling pathways involved in this oncogenic process are however only partially elucidated. Interestingly, SILAC-based quantitative proteomics allow the identification of the whole spectrum of TK substrates and the dynamic of phosphorylation state involved in oncogenic signaling. For example, this approach has highlighted the unsuspected complexity of the oncogenic signaling induced by the TK Src in colorectal cancer (CRC) cells. In this review, we describe a new SILAC-based technology applied to in vivo models of human tumors engrafted in nude mice. This method revealed significant differences between Src-oncogenic signaling of CRC cells in tumors and in culture. Finally, we discuss the interest of SILAC with recently described in vivo proteomic methods and in cancer, including the analysis of oncogenic signaling in tumor progression and the anti-tumoral activity of TK inhibitors in vivo.

Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

New functions of DDR1 collagen receptor in tumor dormancy, immune exclusion and therapeutic resistance.

The tumor microenvironment facilitates cancer progression and therapeutic resistance. Tumor collagens and their architecture play an essential role in this process. However, little is known about the mechanisms by which tumor cells sense and respond to this extracellular matrix environment. Recently, the Discoidin Domain Receptor 1 (DDR1), a collagen receptor and tyrosine kinase has emerged as an important player in this malignant process, although the underlying signaling mechanisms remain unclear. Here, we review new DDR1 functions in tumor dormancy following dissemination, immune exclusion and therapeutic resistance induced by stromal collagens deposition. We also discuss the signaling mechanisms behind these tumor activities and the therapeutic strategies aiming at targeting these collagens-dependent tumor responses.

Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer.

The PEAK family pseudokinases are essential components of tyrosine kinase (TK) pathways that regulate cell growth and adhesion; however, their role in human cancer remains unclear. Here, we report an oncogenic activity of the pseudokinase PEAK2 in colorectal cancer (CRC). Notably, high PRAG1 expression, which encodes PEAK2, was associated with a bad prognosis in CRC patients. Functionally, PEAK2 depletion reduced CRC cell growth and invasion in vitro, while its overexpression increased these transforming effects. PEAK2 depletion also reduced CRC development in nude mice. Mechanistically, PEAK2 expression induced cellular protein tyrosine phosphorylation, despite its catalytic inactivity. Phosphoproteomic analysis identified regulators of cell adhesion and F-actin dynamics as PEAK2 targets. Additionally, PEAK2 was identified as a novel ABL TK activator. In line with this, PEAK2 expression localized at focal adhesions of CRC cells and induced ABL-dependent formation of actin-rich plasma membrane protrusions filopodia that function to drive cell invasion. Interestingly, all these PEAK2 transforming activities were regulated by its main phosphorylation site, Tyr413, which implicates the SRC oncogene. Thus, our results uncover a protumoural function of PEAK2 in CRC and suggest that its deregulation affects adhesive properties of CRC cells to enable cancer progression.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region.

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase.

The PEAK1 and Pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signaling mechanism remains unclear. In this study, we found that, although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, the PEAK3 protein is strongly expressed in human hematopoietic cells and is upregulated in acute myeloid leukemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signaling, independent of growth-factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signaling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2, which promoted PEAK3 tyrosine phosphorylation, its association with GRB2 and ASAP1, and AKT signaling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signaling proteins to promote oncogenesis.

The Tom1L1-clathrin heavy chain complex regulates membrane partitioning of the tyrosine kinase Src required for mitogenic and transforming activities.

Compartmentalization of Src tyrosine kinases (SFK) plays an important role in signal transduction induced by a number of extracellular stimuli. For example, Src mitogenic signaling induced by platelet-derived growth factor (PDGF) is initiated in cholesterol-enriched microdomain caveolae. How this Src subcellular localization is regulated is largely unknown. Here we show that the Tom1L1-clathrin heavy chain (CHC) complex negatively regulates the level of SFK in caveolae needed for the induction of DNA synthesis. Tom1L1 is both an interactor and a substrate of SFK. Intriguingly, it stimulates Src activity without promoting mitogenic signaling. We found that, upon association with CHC, Tom1L1 reduced the level of SFK in caveolae, thereby preventing its association with the PDGF receptor, which is required for the induction of mitogenesis. Similarly, the Tom1L1-CHC complex reduced also the level of oncogenic Src in cholesterol-enriched microdomains, thus affecting both its capacity to induce DNA synthesis and cell transformation. Conversely, Tom1L1, when not associated with CHC, accumulated in caveolae and promoted Src-driven DNA synthesis. We concluded that the Tom1L1-CHC complex defines a novel mechanism involved in negative regulation of mitogenic and transforming signals, by modulating SFK partitioning at the plasma membrane.

Collagen Kinase Receptors as Potential Therapeutic Targets in Metastatic Colon Cancer.

Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide. While surgery can cure patients with early stage CRC, the 5-year survival rate is only 10% for patients with metastatic disease. Therefore, new anti-metastatic therapies are needed for this cancer. Metastatic spread defines the dissemination of cancer cells with tumor-initiating capacities from the primary tumor and their colonization of distinct organs, mainly the liver, for secondary tumor formation. Although the underlying mechanisms are not fully understood, components of the tumor microenvironment have gained strong interest. Among the known metastatic-promoting factors, collagens are extracellular matrix components that are deposited within the tumor, the tumor microenvironment, and at metastatic site(s), and are recognized to play essential roles during metastasis development. Here, we review recent findings on the metastatic role of the collagen receptors Discoidin Domain Receptors 1 and 2 (DDR1 and DDR2) in CRC and discuss the therapeutic value of targeting these receptor tyrosine kinases in this cancer.

Src Family Tyrosine Kinases in Intestinal Homeostasis, Regeneration and Tumorigenesis.

Src, originally identified as an oncogene, is a membrane-anchored tyrosine kinase and the Src family kinase (SFK) prototype. SFKs regulate the signalling induced by a wide range of cell surface receptors leading to epithelial cell growth and adhesion. In the intestine, the SFK members Src, Fyn and Yes regulate epithelial cell proliferation and migration during tissue regeneration and transformation, thus implicating conserved and specific functions. In patients with colon cancer, SFK activity is a marker of poor clinical prognosis and a potent driver of metastasis formation. These tumorigenic activities are linked to SFK capacity to promote the dissemination and tumour-initiating capacities of epithelial tumour cells. However, it is unclear how SFKs promote colon tumour formation and metastatic progression because SFK-encoding genes are unfrequently mutated in human cancer. Here, we review recent findings on SFK signalling during intestinal homeostasis, regeneration and tumorigenesis. We also describe the key nongenetic mechanisms underlying SFK tumour activities in colorectal cancer, and discuss how these mechanisms could be exploited in therapeutic strategies to target SFK signalling in metastatic colon cancer.

The adaptor protein Tom1L1 is a negative regulator of Src mitogenic signaling induced by growth factors.

The Src family of protein-tyrosine kinases (SFK) play important roles in mitogenesis and morphological changes induced by growth factors. The involved substrates are, however, ill defined. Using an antiphosphotyrosine antibody to screen tyrosine-phosphorylated cDNA expression library, we have identified Tom1L1, an adaptor protein of the Tom1 family and a novel substrate and activator of the SFK. Surprisingly, we found that Tom1L1 does not promote DNA synthesis induced by Src. Furthermore, we report that Tom1L1 negatively regulates SFK mitogenic signaling induced by platelet-derived growth factor (PDGF) through modulation of SFK-receptor association: (i) Tom1L1 inhibits DNA synthesis induced by PDGF; (ii) inhibition is overcome by c-myc expression or p53 inactivation, two regulators of SFK mitogenic function; (iii) Src or Fyn coexpression overrides Tom1L1 mitogenic activity; (iv) overexpression of the adaptor reduces Src association with the receptor; and (v) protein inactivation potentiates receptor complex formation, allowing increased SFK activation and DNA synthesis. However, Tom1L1 affects neither DNA synthesis induced by the constitutively active allele SrcY527F nor SFK-regulated actin assembly induced by PDGF. Finally, overexpressed Tom1 and Tom1L2 also associate with Src and affected mitogenic signaling in agreement with some redundancy among members of the Tom1 family. We concluded that Tom1L1 defines a novel mechanism for regulation of SFK mitogenic signaling induced by growth factors.

Cytoplasmic signalling by the c-Abl tyrosine kinase in normal and cancer cells.

c-Abl is a non-receptor tyrosine kinase which is localized both in the nucleus and cytoplasm, and is involved in the regulation of cell growth, survival and morphogenesis. Although c-Abl nuclear function has been extensively studied, recent data also indicate an important role in cytoplasmic signalling through mitogenic and adhesive receptors. Here, we review the mechanisms by which growth factors promote cytoplasmic c-Abl activation and signalling and its function in the induction of DNA synthesis, changes in cell morphology and receptor endocytosis. The importance of de-regulated c-Abl cytoplasmic signalling in solid tumours is also discussed.

Control of Tyrosine Kinase Signalling by Small Adaptors in Colorectal Cancer.

Tyrosine kinases (TKs) phosphorylate proteins on tyrosine residues as an intracellular signalling mechanism to coordinate intestinal epithelial cell communication and fate decision. Deregulation of their activity is ultimately connected with carcinogenesis. In colorectal cancer (CRC), it is still unclear how aberrant TK activities contribute to tumour formation because TK-encoding genes are not frequently mutated in this cancer. In vertebrates, several TKs are under the control of small adaptor proteins with potential important physiopathological roles. For instance, they can exert tumour suppressor functions in human cancer by targeting several components of the oncogenic TK signalling cascades. Here, we review how the Src-like adaptor protein (SLAP) and the suppressor of cytokine signalling (SOCS) adaptor proteins regulate the SRC and the Janus kinase (JAK) oncogenic pathways, respectively, and how their loss of function in the intestinal epithelium may influence tumour formation. We also discuss the potential therapeutic value of these adaptors in CRC.

SHEDding light on the role of Pragmin pseudo-kinases in cancer.

The human kinome comprises more than 50 pseudo-kinases with unclear biological function due to the absence of apparent catalytic activity, and therefore, with presumably little interest for cancer drug discovery. However, it is now acknowledged that several of them, such as Pragmin family members, play roles as important as those of active kinases in human cancer. How these pseudo-kinases promote tumor formation is largely unknown. Recently, independent structural analyses of three Pragmin pseudo-kinases (Pragmin, SGK223, and SGK269/PEAK1) revealed a split helical dimerization (SHED)-based mechanism of action. Additional sequence-structure analysis identified C19orf35 as a new member of the Pragmin family. Based on the results of these molecular studies, we present a unified model on how Pragmin pseudo-kinases may regulate oncogenic signaling, and suggest potential therapeutic strategies to block their tumor activity.

DDR1 inhibition as a new therapeutic strategy for colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) is still a major challenge. Recently, we discovered that nilotinib, an approved treatment for chronic myeloid leukaemia, inhibits invasive and metastatic properties of CRC cells by targeting the kinase activity of receptor for collagens DDR1 (Discoïdin Domain Receptor tyrosine kinase 1), suggesting that nilotinib could be an effective strategy to treat mCRC.

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation.

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1,368 amino acid C terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein-kinase fold devoid of catalytic activity, despite a conserved catalytic lysine (K997). By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein-kinase domain is flanked by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. These results reveal a dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation. Further sequence-structure analysis identified an additional member (C19orf35) of the superfamily of dimeric Pragmin/SgK269/PEAK1 pseudo-kinases.