Uncoupling JAK3 activation induces apoptosis in human lymphoid cancer cells via regulating critical survival pathways

In the current work, we report that specific inhibition of Janus tyrosine kinase (JAK3) via NC1153 induces apoptosis of certain leukemia/lymphoma cell lines. Affymetrix microarray profiling following NC1153 treatment unveiled JAK3 dependent survival modulating pathways (p53, TGF-β, TNFR and ER stress) in Kit225 cells. IL-2 responsive NC1153 target genes were regulated in human JAK3 positive, but not in JAK3 negative lymphoid tumor cells. Moreover, primary lymphoma samples revealed that a number of these genes were reciprocally regulated during disease progression and JAK3 inhibition suggesting that downstream targets of JAK3 could be exploited in the development of novel cancer treatment regimes.     

Gene Expression Analysis

The response of cells to extracellular signals usually requires altered expression of many genes, possibly including several distinct metabolic pathways. In some cases, only a subset of genes involved in such responses are known, which requires techniques to analyze changes in the expression of multiple genes, both known and unknown. Three techniques, two‐dimensional gel electrophoresis, differential display, and gene discovery arrays, provide opportunities for measuring changes in gene expression levels, as well as for identifying novel gene products.     

Gene expression analysis

The response of cells to extracellular signals usually requires altered expression of many genes, possibly including several distinct metabolic pathways. In some cases, only a subset of genes involved in such responses are known, which requires techniques to analyze changes in the expression of multiple genes, both known and unknown. Three techniques, two-dimensional gel electrophoresis, differential display, and gene discovery arrays, provide opportunities for measuring changes in gene expression levels, as well as for identifying novel gene products.     

3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3′-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (∼9.6K genes) and FFPET (∼8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (∼4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.     

Cooperation of JAK/STAT and Notch signaling in the Drosophila foregut

Temporal and spatial regulation of morphogenesis is pivotal to the formation of organs from simple epithelial tubes. In a genetic screen for novel genes controlling cell movement during posterior foregut development, we have identified and molecularly characterized two alleles of the domeless gene which encodes the Drosophila Janus kinase (JAK)/STAT receptor. We demonstrate that mutants for domeless or any other known component of the canonical JAK/STAT signaling pathway display a failure of coordinated cell movement during the development of the proventriculus, a multiply folded organ which is formed by stereotyped cell rearrangements in the posterior foregut. Whereas the JAK/STAT receptor is expressed in all proventricular precursor cells, expression of upd encoding its ligand and of STAT92E, the signal transducer of the pathway, is locally restricted to cells that invaginate during proventriculus development. We demonstrate by analyzing gene expression mediated by a model Notch response element and by studying the expression of the Notch target gene short stop, which encodes a cytoskeletal crosslinker protein, that JAK/STAT signaling is required for the activation of Notch-dependent gene expression in the foregut. Our results provide strong evidence that JAK/STAT and Notch signaling cooperate in the regulation of target genes that control epithelial morphogenesis in the foregut.     

JAK/STAT3-dependent activation of the RalGDS/Ral pathway in M1 mouse myeloid leukemia cells.

The Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral guanine nucleotide dissociation stimulator (RalGDS), a putative effector protein of Ras. Recently, it was proven that Ral regulates c-Src activity and subsequent phosphorylation of its substrate, STAT3. Here, we show that STAT3 inversely regulates activation of Ral through induction of expression of RalGDS. To identify new leukemia inhibitory factor-induced genes, we have performed representational difference analysis using M1 mouse myeloid leukemia cells and cloned RalGDS. The expression of RalGDS and subsequent activation of RalA were clearly suppressed by a dominant negative form of STAT3 and a JAK inhibitor, JAB/SOCS1/SSI-1, indicating that RalGDS/RalA signaling requires the activation of the JAK/STAT3 pathway. An experiment using a Ras inhibitor demonstrated that full activation of RalA also requires activation of Ras. These results suggest a novel cross-talk between JAK/STAT3 and the Ras/RalGDS/Ral signaling pathways through gp130.     

MicroRNAs Are Part of the Regulatory Network that Controls EGF Induced Apoptosis,Including Elements of the JAK/STAT Pathway,in A431 Cells

MiRNAs are known to regulate gene expression and in the context of cancer have been shown to regulate metastasis, cell proliferation and cell death. In this report we describe potential miRNA regulatory roles with respect to induction of cell death by pharmacologic dose of Epidermal Growth Factor (EGF). Our previous work suggested that multiple pathways are involved in the induction of apoptosis, including interferon induced genes, cytokines, cytoskeleton and cell adhesion and TP53 regulated genes. Using miRNA time course expression profiling of EGF treated A431 cells and coupling this to our previous gene expression and proteomic data, we have been able to implicate a number of additional miRNAs in the regulation of apoptosis. Specifically we have linked miR-134, miR-145, miR-146b-5p, miR-432 and miR-494 to the regulation of both apoptotic and anti-apoptotic genes expressed as a function of EGF treatment. Whilst additional miRNAs were differentially expressed, these had the largest number of apoptotic and anti-apoptotic targets. We found 5 miRNAs previously implicated in the regulation of apoptosis and our results indicate that an additional 20 miRNAs are likely to be involved based on their correlated expression with targets. Certain targets were linked to multiple miRNAs, including PEG10, BTG1, ID1, IL32 and NCF2. Some miRNAs that target the interferon pathway were found to be down regulated, consistent with a novel layer of regulation of interferon pathway components downstream of JAK/STAT. We have significantly expanded the repertoire of miRNAs that may regulate apoptosis in cancer cells as a result of this work.