Examination of the roles of the AP-1 transcription factors, JunB and c-Jun, in the pathobiology of Anaplastic Lymphoma Kinase-positive, Anaplastic Large Cell Lymphoma (ALK+ ALCL)

  • Author / Creator
    Wu, Zuoqiao
  • Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive T cell lymphoma that is characterized by chromosomal translocations involving the gene encoding for the ALK tyrosine kinase. The ALK fusion proteins generated through these translocations initiate downstream signalling pathways and contribute to the pathobiology of ALK+ ALCL. Signalling by NPM-ALK, the most common ALK fusion protein, elevates the expression and/or constitutively activates the activator protein-1 (AP-1) transcription factors, JunB and c-Jun. In this thesis, I investigated the role that JunB and c-Jun play in the pathobiology of ALK+ ALCL.To examine the role of JunB and c-Jun in ALK+ ALCL, the expression of the two AP-1 proteins was knocked-down stably in ALK+ ALCL cell lines with short-hairpin RNAs (shRNA)s. I found that knock-down of JunB resulted in a reduced growth rate characterized by defect in G0/G1 cell cycle progression in the majority of the cell lines examined. In contrast, knock-down of c-Jun in multiple cell lines resulted in no observable effect on proliferation.To further investigate the function of JunB in the pathobiology of ALK+ ALCL, microarray experiments were performed to compare the gene expression between control shRNA-expressing and JunB shRNA-expressing Karpas 299 cells, an ALK+ ALCL cell line. When JunB was knocked-down in Karpas 299 cells, expression of 678 genes (549 up-regulated and 132 down-regulated) were altered by a greater than 2-fold change. KEGG pathway annotation analysis revealed that “natural killer cell mediated cytotoxicity” as the most statistically significant category amongst the functional pathways that showed up in the analysis. Within this functional category, I confirmed thatIIIseveral ligands for NK activating receptors were up-regulated at the mRNA and protein levels in JunB knock-down cells. My data further suggested that the up-regulation of these ligands could be partially due to epigenetic changes. More importantly, my data suggested that the up-regulation of these ligands made one ALK+ ALCL cell line more susceptible to NK-mediated killing.To further identify the transcriptional targets of JunB and c-Jun, ChIP-seq was performed in Karpas 299 cells with anti-JunB and anti-c-Jun antibodies. I found a large number of genes were associated with JunB or/and c-Jun intervals, which was consistent with the fact that AP-1 sites are abundant in the genome. More genes were associated with JunB intervals alone or both JunB and c-Jun intervals, and a small number of genes were associated with c-Jun intervals alone. This suggested that JunB and c-Jun could regulate common genes, but JunB may regulate many more genes than c-Jun. FAM129B was identified as a potential direct transcriptional target of JunB and c-Jun because it was associated with JunB and c-Jun intervals at the putative promoter based on the ChIP-seq data. Knock-down of FAM129B resulted in a growth defect and increased sub-G0/G1 populations in ALK+ ALCL cell lines. Moreover, FAM129B electrophoretic mobility was dependent on NPM-ALK activity, suggesting that NPM-ALK may promote the phosphorylation of FAM129B.In summary, I showed that JunB played a critical role in promoting proliferation in ALK+ ALCL, but this function was not shared with the related transcription factor, c- Jun. Secondly, I addressed the potential role for JunB in protecting ALK+ALCL tumour cells from immune surveillance. Thirdly, I identified a novel potential JunB and c-Jun transcriptional target that could contribute to growth and survival of ALK+ ALCL cells.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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