Suppressive Functions of ATF2/7 in oncogenic Ras mediated cellular transformation
In tumorigenesis, ATF2 can exert pro-tumorigenic but also anti-tumorigenic activities depending on the tumour type (Gozdecka and Breitwieser, 2012). Therefore one focus of our research has been to decipher these context dependent activities. In a current project we adapted a mouse model of hepatocellular carcinoma (HCC), whereby oncogenic HRas transformed hepatoblasts, proficient or deficient in ATF2/7 functions, are transplanted orthotopically and develop primary liver tumours. As a result, we found that ATF2/7 double mutant hepatoblasts show a significantly stronger tendency to develop into HCC in recipient livers compared to ATF2 active controls. We also found that active ATF2 induces cell death in transformed hepatoblasts in culture and reduces colony formation in soft agar. These activities appear to be dependent on the activation of an upstream kinase (JNK) because expression of a phosphorylation deficient form of ATF2 (ATF2-AA) fails to rescue the ATF2 loss of function mutant. Furthermore, we find that expression of active JNK had similar suppressive activities during oncogenic transformation of hepatoblasts and that these activities strictly require ATF2/7. Therefore, tumour suppressive functions involving JNK mediated pathways are at least in part dependent on ATF2/7 transcriptional activities. Using microarray analysis we identified ATF2 target genes that may be involved in tumour suppression. In meta-analysis of tumour expression data we find that down-regulation of many ATF2/7 targets correlates with tumour status.
ATF2/7 roles in Myc transformed B cells and B lymphoma
In samples of human B cell lymphoma cell lines, we found that ATF2 as well as JNK MAP kinase are significantly up-regulated compared to normal human B cell lines. This was particularly the case in Burkitt’s lymphoma and other lymphoma types, typically associated with the activation of the c-Myc transcription factor. To analyse the potential interaction between activated c-Myc and the JNK-ATF2 signalling pathway we developed a mouse model in which ATF2 and 7 were specifically deleted in the B cell compartment and tested this in the presence of B cell specific overexpression of c-Myc (Eµ-Myc). As with the human B lymphoma samples, we found that mouse lymphomas induced by Myc overexpression also showed strongly enhanced activation of JNK as well as of ATF2. Furthermore, B cell specific loss of ATF2/7 resulted in a significant acceleration of Eµ-Myc induced lymphoma onset suggesting that the JNK-ATF2/7 pathway is engaged in tumour suppressive activities in a Myc overexpression context.
To further characterise these activities we derived cell lines from Myc induced primary lymphomas and induced the deletion of ATF2 in vitro using Cre/loxP mediated recombination. Here we found that while loss of ATF2/7 in B lymphoma cells did not affect growth in culture, they showed remarkably reduced levels of spontaneous apoptosis as well as reduced levels of apoptosis induced by growth factor withdrawal or treatment with genotoxic drugs, e.g. doxorubicin or mitoxantrone. It has previously been shown that while forced Myc expression induces proliferation of lymphoma and other cells it also sensitises cells to stress induced apoptosis. Our findings therefore suggest that JNK and ATF2/7 may be at least partially responsible for the apoptotic arm of Myc activities and that this could underlie the observed tumour suppressive activities of ATF2/7 in the mouse B lymphoma model. In an analysis of ATF2/7 dependent transcription regulation we showed that transcriptional targets include other AP-1 factors, including c-Jun and ATF3 as well as at least one member of BH3 domain containing apoptotic regulators, Hrk (Walczynski J et al. Oncogene 2013).
Oncogenic Ras regulated microRNAs involved in suppression of transformation and drug induced apoptosis
Over the past few years it has become apparent that non coding RNAs display important functions in the regulation of gene expression and modulation of signalling pathways. In an approach to identify new mechanisms involved in oncogenic Ras-mediated signalling, we identified a number of microRNAs that were specifically down regulated in their expression in response to activated HRas. Upon further characterisation we found that a number of these miRs, including miR99 and miR335 are involved in the suppression of Hras mediated transformation, for example by reducing colony formation in soft agar. Furthermore, ectopic expression of miR335 leads to enhanced apoptosis in response to genotoxic drugs such as cisplatin. To evaluate this role for miR335 in a clinically relevant setting we analysed ovarian tumour cell lines for the expression of miR335 and found that while a number of these show strong resistance to cisplatin induced cell death they also have very low expression of miR335. In contrast re-expression of miR335 strongly re-sensitises cisplatin resistant cell lines to the cisplatin-induced apoptosis. A typical mode of evasion to drug sensitivity by tumour cells is by modifications in the methylation status of the genome. Interestingly, we found that treatment of drug resistant cells with demethylating agents led to enhanced expression of miR335 in correlation with their increased sensitisation to cisplatin treatment. We therefore aim to further test the possibility that at least one mechanism of acquired drug resistance in tumours is by silencing the expression of microRNAs including miR335. We are also exploring the targets of mir335 involved in cisplatin induced cellular responses.
Figure 1: Apoptotic (Caspase 3) staining of Myc induced mouse lymphomas.