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.

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.

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.

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.

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.

Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

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.

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.

Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS-independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1-mediated BCR phosphorylation on Tyr177, which is important for maintaining ß-catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient-derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.

Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element.

Serum levels of HDL are inversely correlated with the risk of coronary heart disease. The anti-atherogenic effect of HDL is partially mediated by its major protein constituent apoA-I. In this study, we identify bile acids that are activators of the nuclear receptor farnesoid X receptor (FXR) as negative regulators of human apoA-I expression. Intrahepatocellular accumulation of bile acids, as seen in patients with progressive familial intrahepatic cholestasis and biliary atresia, was associated with diminished apoA-I serum levels. In human apoA-I transgenic mice, treatment with the FXR agonist taurocholic acid strongly decreased serum concentrations and liver mRNA levels of human apoA-I, which was associated with reduced serum HDL levels. Incubation of human primary hepatocytes and hepatoblastoma HepG2 cells with bile acids resulted in a dose-dependent downregulation of apoA-I expression. Promoter mutation analysis and gel-shift experiments in HepG2 cells demonstrated that bile acid-activated FXR decreases human apoA-I promoter activity by a negative FXR response element mapped to the C site. FXR bound this site and repressed transcription in a manner independent of retinoid X receptor. The nonsteroidal synthetic FXR agonist GW4064 likewise decreased apoA-I mRNA levels and promoter activity in HepG2 cells.

Therapeutic Activity of Anti-AXL Antibody against Triple-Negative Breast Cancer Patient-Derived Xenografts and Metastasis.

Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation. Clin Cancer Res; 23(11); 2806-16. ©2016 AACR.

ABL tyrosine kinase inhibition variable effects on the invasive properties of different triple negative breast cancer cell lines.

The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN) breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.

The farnesoid X receptor induces very low density lipoprotein receptor gene expression.

The farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). In response to ligand-binding, FXR regulates many genes involved in BA, lipid, and lipoprotein metabolism. To identify new FXR target genes, microarray technology was used to profile total RNA extracted from HepG2 cells treated with the natural FXR agonist chenodeoxycholic acid (CDCA). Interestingly, a significant increase of transcript level of the very low density lipoprotein receptor (VLDLR) was observed. Our data, resulting from selective FXR activation, FXR RNA silencing and FXR-deficient mice, clearly demonstrate that BAs up-regulate VLDLR transcript levels via a FXR-dependent mechanism in vitro in human and in vivo in mouse liver cells.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2.

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

Oncogenic signaling by tyrosine kinases of the SRC family in advanced colorectal cancer.

The non-receptor tyrosine kinases of the SRC family (SFK) play important roles in signal transduction induced by a large variety of extracellular stimuli, including growth factors and Integrins. When deregulated, SFKs show oncogenic activity, as originally reported for v-Src, the transforming product of the avian retrovirus RSV, and then, in many human cancers, particularly colorectal cancer (CRC). In CRC, SFK deregulation largely occurs in the absence of mutations of the corresponding genes, but the underlying molecular mechanisms involved are still unclear. In addition to a role in early tumor progression, SFK deregulation may also be important in advanced CRC, as suggested by the association between increased SFK activity and poor clinical outcome. However, SFK contribution to CRC metastasis formation is still poorly documented. Here, we will review recent findings that broaden our understanding of the mechanisms underlying SFK deregulation and signaling in advanced CRC. We will also discuss the implication of these observations for SFK-based therapy in metastatic CRC.