Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation

Abstract

Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.

Figure 1

Transcript profile analysis of Upd-induced JAK/STAT activation. (A) Upd is able to stimulate a luciferase JAK/STAT pathway reporter construct (Müller et al, 2005). (B–D) Quantitative PCR showing socs36E mRNA levels in Kc₁₆₇ cells after either Upd stimulation (B,C) or 72 h after Hop^TumL transfection (D). (E–G) Plots of genes expressed at the indicated time points showing the log₂ ratio for each gene as a function of the mean signal intensity. Differentially expressed genes were identified by calculating intensity-dependent z-scores and filtered to remove those differing by less than two standard deviations from the mean. (H) The number of genes either upregulated or downregulated at the 2 h, 4 h and 10 h time points. (I) Venn diagram showing the distribution of upregulated and downregulated genes at the 2 h, 4 h and 10h time points. The 11 loci upregulated at all time points are listed. Hop, Hopscotch; JAK, Janus kinase; STAT, signal transducer and activator of transcription; Upd, Unpaired.

Figure 2

Clustering, gene ontology and binding-site analysis of putative JAK/STAT target genes. (A) Hierarchical cluster analysis of 1,168 Upd-regulated genes using log₂ ratios. The blue tiles represent downregulated genes and red tiles indicate upregulated genes. Six main clusters were identified. (B) The functional enrichment of the 1,168 Upd-regulated genes according to gene ontology terms. (C) The average number of potential 3n and 4n STAT92E-binding sites present in the upstream region of positively regulated genes at each time point, compared with the number of sites in a randomly selected ‘control' group of genes. ^*P<0.05, ^**P<0.005, ^***P<0.0005. JAK, Janus kinase; STAT, signal transducer and activator of transcription; Upd, Unpaired.

Figure 3

In vivo characterization of JAK/STAT targets. (A) The third-instar lymph gland showing the domain of p[G5] expression (green) in the central MZ. DNA is shown in magenta. (B,C) Adult flies of the specified genotypes carrying the gain-of-function hop^TumL allele contain multiple, large black tumours (indicated by arrows) when a control shRNA targeting rhodopsin 4 (rh4) is expressed in the MZ by the p[G5] driver (B). Flies in which the pathway effector CG13559 is knocked down contain fewer, smaller tumours (C). The TI (D) and percentage of adults with tumours of the indicated size (E) are shown for the specified genotypes, which either overexpress or knock down socs36E. Overexpression using UAS-SOCS36E-GFP flies showed significant reduction in TI as compared with that in the control (D). (F) The TI of flies of the indicated genotype expressing shRNAs targeting 21 potential effectors as well as rh4 and white as controls. The bar indicates the link between baz misexpression and w[1118] used as the control for this cross. (G,H) Representative differential interference contrast image of haemocytes from hop^TumL larvae of the indicated genotypes showing reduction in circulating cells present after UAS-baz overexpression. (I) Fold change in the expression of selected homologues of Drosophila genes in HeLa cells after stimulation by the gain-of-function JAK2 V617F mutation. For all graphs, error bars represent standard error. ^*P<0.05, ^**P<0.01, ^***P<0.001. baz, bazooka; GFP, green fluorescent protein; hop, Hopscotch; JAK, Janus kinase; MZ, medullary zone; shRNA, short-hairpin RNA; STAT, signal transducer and activator of transcription; TI, tumour index.