Suppressor of cytokine signaling 1 modulates invasion and metastatic potential of colorectal cancer cells.

Suppressor of cytokine signaling (SOCS) 1 is an inducible negative regulator of cytokine signaling but its role in human cancer is not completely established. Here we report that, while SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas, its level decreases during progression of colon adenocarcinomas, the lowest level being found in the most aggressive stage and least differentiated carcinomas. Forced expression of SOCS1 in metastatic colorectal SW620 cells reverses many characteristics of Epithelial-Mesenchymal Transition (EMT), as highlighted by the disappearance of the transcription factor ZEB1 and the mesenchymal form of p120ctn and the re-expression of E-cadherin. Furthermore, miRNA profiling indicated that SOCS1 also up-regulates the expression of the mir-200 family of miRNAs, which can promote the mesenchymal-epithelial transition and reduce tumor cell migration. Accordingly, overexpression of SOCS1 induced cell morphology changes and dramatically reduced tumor cell invasion in vitro. When injected in nude mice, SOCS1-expressing SW620 cells induced metastases in a smaller number of animals than parental SW620 cells, and did not generate any adrenal gland or bone metastasis. Overall, our results suggest that SOCS1 controls metastatic progression of colorectal tumors by preventing the mesenchymal-epithelial transition (MET), including E-cadherin expression. This pathway may be associated with survival to colorectal cancer by reducing the capacity of generating metastases.

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.

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation.

Human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. HEF1 is present at focal adhesions and is involved in integrin signalling mediating cytoskeleton reorganization associated with cell migration, adhesion or apoptosis. HEF1 functions are regulated in part by phosphorylation on tyrosine residues. HEF1 is also phosphorylated on serines/threonines leading to two isoforms refered to as p105 and p115. In most cases, the serine/threonine kinase(s) responsible for HEF1 phosphorylation have not been identified. In the present study, we have investigated HEF1 ser/thr phosphorylation. In the HCT-116 cell line transiently overexpressing Flag-HEF1 we showed that Hesperadin, a synthetic indolinone displaying antiproliferative effect and described as an inhibitor of various kinases including Aurora-B, prevented HEF1 phosphorylation induced by the ser/thr phosphatase PP2A inhibitor: okadaic acid (OA). In addition we showed that conversion of endogenous HEF1 p105 to p115 in HaCaT cells was prevented upon treatment with Hesperadin, resulting in accumulation of p105HEF1. We also identified serine 369 as the target site of phosphorylation by this Hesperadin-inhibited kinase in HCT-116. Finally, we provide evidence that phosphorylation on serine 369 but not phosphorylation on serine 296, triggers HEF1 degradation by the proteasomal machinery. These data suggest that conversion of p105 to p115 results from a ser-369-dependent phosphorylation mediated by an Hesperadin-sensitive kinase and regulates the half-life of HEF1.

JAK/STAT signal transduction: regulators and implication in hematological malignancies.

Signal transducers and activators of transcription (STATs) comprise a family of several transcription factors that are activated by a variety of cytokines, hormones and growth factors. STATs are activated through tyrosine phosphorylation, mainly by JAK kinases, which lead to their dimerization, nuclear translocation and regulation of target genes expression. Stringent mechanisms of signal attenuation are essential for insuring appropriate, controlled cellular responses. Among them phosphotyrosine phosphatases (SHPs, CD45, PTP1B/TC-PTP), protein inhibitors of activated STATs (PIAS) and suppressors of cytokine signaling (SOCS) inhibit specific and distinct aspects of cytokine signal transduction. SOCS proteins bind through their SH2 domain to phosphotyrosine residues in either cytokine receptors or JAK and thus can suppress cytokine signaling. Many recent findings indicate that SOCS proteins act, in addition, as adaptors that regulate the turnover of certain substrates by interacting with and activating an E3 ubiquitin ligase. Thus, SOCS proteins act as negative regulators of JAK/STAT pathways and may represent tumour suppressor genes. The discovery of oncogenic partner in this signaling pathway, more especially in diverse hematologic malignancies support a prominent role of deregulated pathways in the pathogenesis of diseases. Fusion proteins implicating the JH1 domain of JAK2 (TEL-JAK2, BCR-JAK2), leading to deregulated activity of JAK2, have been described as the result of translocation. Somatic point mutation in JH2 domain of JAK2 (JAK2V617F), leading also to constitutive tyrosine phosphorylation of JAK2 and its downstream effectors was reported in myeloproliferative disorders. Furthermore, silencing of socs-1 and shp-1 expression by gene methylation is observed in some cancer cells.

Interleukin-4 downregulates TNFalpha-induced IL-8 production in keratinocytes.

Interleukin (IL)-8 is a CXC chemokine induced by pro-inflammatory cytokines such as TNFalpha, IL-1beta and IL-6 in different cell types including keratinocytes. IL-4 regulation of TNFalpha-induced IL-8 expression is cell-type specific. In this study, we show that in the keratinocyte cell line HaCaT, IL-4 decreases TNFalpha-induced IL-8 mRNA expression. We then investigated the mechanism of IL-4 effect and showed that IL-4 downregulates TNFalpha-induced IL-8 promoter activity in luciferase reporter assays. Moreover, overexpression of either the endogenous JAK inhibitor SOCS-1 or a dominant negative form of the STAT6 transcription factor (STAT6DeltaC) interferes with the IL-4 inhibitory effect on IL-8 promoter. Finally we demonstrate, using a NF-kappaB-dependent promoter luciferase construct that IL-4 interferes, at least in part, with NF-kappaB transcriptional activity. Overall our results suggest that IL-4 regulates TNFalpha-induced IL-8 expression at a transcriptional level and this mechanism involves STAT6 and NF-kappaB transcription factors.

STAT6 and Ets-1 form a stable complex that modulates Socs-1 expression by interleukin-4 in keratinocytes.

Supressor of cytokine signaling (SOCS)-1 is selectively and rapidly induced by appropriate agonists and modulates cytokine responses by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observation that interleukin (IL)-4 up-regulates Socs-1 in the keratinocyte HaCaT cell line, we investigated which sequences of the 5'-Socs-1 gene are responsive to IL-4. We therefore have cloned the 5'-flanking region of this gene, and by promoter analysis we identified a functional IL-4-responsive element located at nucleotide (-684/-570) upstream from the transcription initiation site, whose presence and integrity are necessary to ensure IL-4 responsiveness. This element contains three STAT6 and one Ets consensus binding sequences of which specific mutations abolished IL-4 responsiveness either partially or totally. We also report that Ets-1 physically interacted with STAT6. Exogenous expression of Ets-1 in conjunction with STAT6 activation strongly inhibited expression of a Socs-1 promoter-luciferase reporter. Collectively, our data demonstrated the involvement of STAT6 and Ets, via a composite DNA element, in the IL-4 regulation of Socs-1 gene expression in keratinocytes.

Functional characterization of IL-13 receptor alpha2 gene promoter: a critical role of the transcription factor STAT6 for regulated expression.

Interleukin (IL)-4 and IL-13 are two structurally and functionally related cytokines that have overlapping but also distinct biological activities. One of the components of the IL-13 receptor, the alpha2 chain (IL-13Ralpha2), has been reported to downregulate the cell responsiveness to IL-13, without affecting IL-4 signaling. Here, we report that TNFalpha synergizes with either IL-4 or IL-13 in inducing the IL-13Ralpha2 chain at both the mRNA and protein levels in the HaCaT human keratinocyte cell line. Further studies by 5'RACE identified as yet undescribed exonic sequences of the IL-13Ralpha2 5'UTR, provided evidence for the expression of alternatively spliced IL-13Ralpha2 transcripts and defined the transcription start of the IL-13Ralpha2 gene. A 1.5 kb region upstream of the first exon of the IL-13Ralpha2 gene displayed basal promoter activity when inserted in a reporter plasmid and transiently transfected in HaCaT cells. This promoter activity was further increased in response to IL-4 and IL-13. Furthermore, by electrophoretic mobility shift assay and site-directed mutagenesis, we showed that the IL-4/IL-13-induced promoter activity depended upon a positively acting STAT6 response element. Finally, TNFalpha was shown to potentiate IL-4/IL-13-induced IL-13Ralpha2 promoter activity when the same reporter construct was studied in stably but not in transiently transfected cells. These results suggest that the synergistic effect of TNFalpha on IL-4/IL-13-induced IL-13Ralpha2 expression is dependent upon chromatin re-modeling events.