A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial–mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer

Exploring the genome-wide impact of transcription factor AP-1 in breast cancer

AP-1 plays crucial roles in a wide range of cellular processes in breast cancer. Through the dimeric basic leucine zipper (bZIP) domain, the mammalian AP-1 proteins bind to DNA and form homodimers or heterodimers from the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra1, Fra2), ATF and MAF family members. AP-1 is involved in several signal transduction pathways to control physiological and pathological processes, such as oncogenesis, metastasis and apoptosis. However, the mechanistic aspects of the modulatory effect of AP-1 in breast cancer are still not fully understood. Thus, to explore genome-wide transcriptional regulatory networks of the transcription factor AP-1 in breast cancer may help to identify novel strategies to develop new therapies. In Paper I, we established that AP-1 participates in estrogen-dependent gene expression and proliferation programs in breast cancer cells. In addition, we identified PKIB (cAMP-dependent protein kinase inhibitor-β) as a novel ERα/AP-1 target molecule, which is required for breast cancer cell growth. In Paper II, we observed, by analyzing publically available datasets, that AP-1 is expressed at high levels in basal-like breast cancers and associated with poor clinical outcome. High level expression of AP-1 was also found in triple-negative breast cancer (TNBC) cell lines as determined by Western blot analysis and qPCR. Using cistrome and transcriptome analyses to investigate the signaling networks of AP-1 in TNBC cells, we identified that about 15% of AP-1 binding sites are located in the proximal 5’ region of the nearest gene. Gene expression profiling analysis identified differential expression of 419 and 690 genes upon knockdown of Fra-1 and c-Jun, respectively. Among these genes, 222 genes which were regulated by both Fra-1 and c-Jun were associated with cytokine-mediated signaling, type I interferon-mediated signaling, chemotaxis, cell adhesion, immune response, cell junction assembly, adherens junction organization and inflammatory response. Moreover, we found that proliferative phenotypes of TNBC cells were inhibited upon depletion of AP-1. In addition, silencing of AP-1 reduced the invasion ability both in vitro and in vivo. We further showed that AP-1 activation, downstream of the PI3K/Akt and MAPK/ERK pathways, repressed expression of E-cadherin by transcriptional upregulation of ZEB2. In Paper III, we demonstrated that TNFα activated both the PI3K/Akt and MAPK/ERK signaling pathways to induce epithelial-mesenchymal transition (EMT) in TNBC cells via activation of AP-1 signaling and increased expression of the EMT regulator ZEB2. Based on published data on spliced transcripts, two alternatively spliced 5’UTR isoforms of the ZEB2 gene were found to be expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrated that AP-1, when activated by TNFα bound to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. In Paper IV, We defined that c-Jun regulated nearly a third of the TNFα-elicited transcriptome. Expression of a c-Jun-regulated pro-invasion gene set was shown to be strongly associated with clinical outcomes in TNBCs. We demonstrated that c-Jun drives TNFα-mediated TNBC malignant characteristics by transcriptional regulation of Ninj1. As exemplified by the c-Jun bound CXC chemokine genes clustered on chromosome 4, we demonstrated that NF-κB might be a pioneer factor and was required for the regulation of TNFα-inducible inflammatory genes, whereas c-Jun had little effect on TNFα-inducible inflammatory genes. In conclusion, our studies give additional insights into the molecular mechanisms of AP-1 in relation to breast cancer cellular processes. We suggest that inhibition of AP-1 could be a new therapeutic strategy for treatment of breast cancer, especially TNBC

Novel approaches for evaluating brassica germplasm for insect resistance

Brassica crops are grown worldwide for food, oil, medicinal and crop rotation properties. They suffer from insect pests which cause large yield and economic losses. Application of insecticides is the preferred way of dealing with insect problems, but it is not only hazardous to the environment, it also affects humans as the chemicals easily get incorporated into the food chain. As a result, new more resistant varieties are urgently needed to meet the demand of growing populations. A set of 200 accessions were classified as resistant (non-preferred) or susceptible (preferred) in response to cabbage aphid feeding in the field. Fifteen accessions were further assessed to characterize and identify the level and location of resistance factors by investigating feeding behaviour of cabbage aphid using the Electrical Penetration Graph (EPG) technique. The feeding behaviour assessment revealed the presence of interspecific & intraspecific variation and presence of resistance factors at multiple levels. The transcriptional response of these accessions under presence and absence of aphid feeding for 24h showed that gene expression is highly regulated in response to aphid feeding. Gene ontology (GO) enrichment study helped identify strong candidate genes for aphid resistance. In addition to this, the gene expression differences between CWR and landraces indicated adaptations of landraces during the process of domestication. Lastly, Gene expression data was used to develop models to predict insect resistance status. In conclusion, the combination of EPG and transcriptomics provides an opportunity to assess brassica germplasm for further research into defence mechanisms of cabbage aphids