Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.

in Data in Brief (2016), (9), 828-838

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot ... [more ▼]

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so. [less ▲]

Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3

in Cellular Signalling (2016), 28(8), 810-25

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression ... [more ▼]

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆ IBB) deletion mutants of importin-α5, as well as ∆ IBB-α3 and ∆ IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition. [less ▲]

What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue.

in PLoS ONE (2016), 11

Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the ... [more ▼]

Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. [less ▲]

SOCS2: physiological and pathological functions.

in Frontiers in Bioscience (Elite Edition) (2016), 8

Suppressors of cytokine signalling (SOCS) proteins are modulators of cytokine and growth factor signalling whose aberrant regulation has been linked to a variety of inflammatory and neoplastic diseases ... [more ▼]

Suppressors of cytokine signalling (SOCS) proteins are modulators of cytokine and growth factor signalling whose aberrant regulation has been linked to a variety of inflammatory and neoplastic diseases. SOCS proteins are able to act as substrate-recruiting component of E3-ubiquitin ligase complexes and target interacting proteins for degradation. At least some of the family members can also directly inhibit tyrosine kinases such as Janus Kinases (JAK). The most studied family members, CIS, SOCS1, SOCS2 and SOCS3 are important regulators of the JAK-STAT pathway. Here, we focus on SOCS2 and review its biological function as well as its implication in pathological processes. Furthermore, we take advantage of the known crystal structures of SOCS2 to discuss the potential effects of a selection of SOCS2 mutations that were identified in tumour tissues. [less ▲]

The oncogenic FIP1L1-PDGFRalpha fusion protein displays skewed signaling properties compared to its wild-type PDGFRalpha counterpart.

in JAK-STAT (2015), 4(1), 1062596

Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate ... [more ▼]

Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate that the oncogenic FIP1L1-PDGFRalpha kinase exhibits a significantly different signaling pattern compared to its PDGFRalpha wild type counterpart. Interestingly, the activation of primarily membrane-based signal transduction processes (such as PI3-kinase- and MAP-kinase- pathways) is remarkably shifted toward a prominent activation of STAT factors. This diverging signaling pattern compared to classical PDGF-receptor signaling is partially coupled to the aberrant cytoplasmic localization of the oncogene, since membrane targeting of FIP1L1-PDGFRalpha restores activation of MAPK- and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRalpha does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRalpha in more detail and found that STAT5 activation does not involve an SH2-domain-mediated binding mechanism. We thus demonstrate that STAT5 activation occurs via a non-canonical activation mechanism in which STAT5 may be subject to a direct phosphorylation by FIP1L1-PDGFRalpha. [less ▲]

Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer.

in British journal of cancer (2014), 111(4), 726-35

BACKGROUND: Over the past years, some members of the family of suppressor of cytokine signalling (SOCS) proteins have emerged as potential tumour suppressors. This study aimed at investigating the ... [more ▼]

BACKGROUND: Over the past years, some members of the family of suppressor of cytokine signalling (SOCS) proteins have emerged as potential tumour suppressors. This study aimed at investigating the clinical significance of SOCS proteins in colorectal carcinoma (CRC). METHODS: We integrated publicly available microarray expression data on CRC in humans, analysed the expression pattern of SOCSs and assessed the predictive power of SOCS2 and SOCS6 for diagnostic purposes by generating receiver operating characteristic curves. Using laser microdissected patient material we assessed SOCS expression on RNA and protein levels as well as their methylation status in an independent CRC patient cohort. Finally, we investigated the prognostic value of SOCS2 and SOCS6. RESULTS: The meta-analysis as well as the independent patient cohort analysis reveal a stage-independent downregulation of SOCS2 and SOCS6 and identify both molecules as diagnostic biomarkers for CRC. We demonstrate a different methylation pattern within the SOCS2 promoter between tumour tissue and normal control tissue in 25% of CRC patients. Furthermore, early CRC stage patients with low expression of SOCS2 display significantly shorter disease-free survival. CONCLUSIONS: Our data offers evidence that SOCS2 and SOCS6 levels are reduced in CRC and may serve as diagnostic biomarkers for CRC patients. [less ▲]

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells.

in JAK-STAT (2013), 2(3), 24574

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation ... [more ▼]

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. [less ▲]

Detection of activated STAT species using electrophoretic mobility shift assay (EMSA) and potential pitfalls arising from the use of detergents.

in Methods in Molecular Biology (2013), 967

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo ... [more ▼]

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo- and heterodimers and show how the preparation of the extracts can influence the distribution of the STAT species observed in the EMSA. We show that detergents can massively influence the STAT dimer distribution. Although it is unclear whether they primarily interfere with STAT DNA binding and/or whether they break up or further oligomerize STATs, the observation may also have an impact on the results of other techniques performed with detergent-containing cell lysates (e.g., coimmunoprecipitations of STATs with other proteins). [less ▲]

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases.

in Journal of Cellular and Molecular Medicine (2010), 14(3), 504-27

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase ... [more ▼]

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. [less ▲]

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.

in Oncogene (2009), 28(34), 3069-80

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS ... [more ▼]

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia. [less ▲]