Recent Progress 2015
The stress activated MAP kinase pathway comprises a multi-tiered signalling cascade involved in a wide range of biological activities. At its heart lie two distinct kinase families, JNK and p38, that are activated not only by cellular stress and inflammation, but also by growth factors and cytokines, and function in the regulation of proliferation, differentiation, cellular senescence and apoptosis. They exert this through the activation of a multitude of substrates ranging from transcription factors, including c-Jun, ATF2, and p53, to mitochondrial apoptotic regulators, including Bcl-2 and Bim. Consequently, in the context of cancer, JNK and p38 have been shown to be critical in oncogenic transformation, tumour metastasis, as well as tumour-stroma interactions.
Not surprisingly, given the multitude of substrates, the roles of kinase families are complex and often seemingly contradictory. While a lot of these functions for JNK dependent signalling has been delineated using cellular and genetic model systems, recent advances in cancer genome sequencing have highlighted the high frequency at which components of stress signalling pathways are deregulated, or mutated in various tumour types including breast, prostate, endometrial and ovarian cancer. The nature of these mutations suggests that the pathway is aberrantly activated in some types of cancer, but to a far greater extent, inactivated in many other types. Thus, stress signalling pathways are thought to be pro-oncogenic or tumour suppressive depending on the cancer context.
ATF2 and ATF7 in development and tumour initiation
One focus of research in the Cell Regulation group has been the AP-1 transcription factors ATF2 and ATF7, which are effector substrates of both JNK and p38 (reviewed in Gozdecka and Breitwieser, 2012). Our research showed that ATF2/7 have essential functions during development of the embryonic liver and that this is dependent on its activation by the SAPK signalling cascade (Breitwieser et al., 2007). This work, as well as a further project describing the deficiencies in mouse brain development (Ackermann et al., 2011) in the absence of functional ATF2/7, uncovered a role for these transcription factors in a negative feedback loop to regulate the potentially hazardous activities of its upstream acting stress kinases.
Figure 1. Loss of ATF2 and 7 sensitises to oncogenic transformation in mouse liver cells. ATF2-wt/7-ko or ATF2/7 double knockout hepatoblasts were transformed with oncogenic HRas and reintroduced into mice by orthotopic injection. Resulting liver tumours were significantly increased in number in the absence of functional ATF2 (arrows in right panel).
High levels of phospho-ATF2 have been detected in both human melanoma and prostate carcinoma samples, and a role for ATF2 in driving progression of these tumours has been suggested in the literature. Conversely, low levels of ATF2 expression in human breast tumours have also been reported. Experimental analysis using mouse tumour models carried out by ourselves, as well as by other research groups, has uncovered diverse roles for ATF2 in tumourigenesis. Accordingly, ATF2 was shown to contribute to melanoma development through its role in melanocyte-specific gene activation (Shah et al, 2010). In contrast, ATF2-deficient mice are sensitised to carcinogen-induced skin tumourigenesis underlining the tumour context dependent activities of the stress signalling pathway. In a mouse model of Myc oncogene-induced B-cell lymphoma development, we showed that ATF2 deficiency results in accelerated disease onset. Further in vitro analysis revealed that ATF2 responds to oncogene-induced cellular stress by inducing programmed cell death (Walczynski et al., 2013).
ATF2 drives transcriptional programmes in tumour suppression
A recent research project has focused on the role of components of the SAPK pathway in hepatocellular carcinoma (Gozdecka et al, 2014). Here, we demonstrated that JNK dependent activation of ATF2 is critical in blocking the oncogenic transformation of hepatocyte precursors (hepatoblasts) as well as in suppressing their tumourigenicity after orthotopic transplantation into recipient mouse livers. In addition, we defined a JNK and ATF2 dependent transcriptional programme that acts in a tumour suppressive manner. Further analysis revealed that this programme is frequently found inactivated or genetically altered in a variety of human tumours types, including breast, brain, colorectal and lung carcinoma. This analysis therefore confirmed that the experimental tumour models reflect human cancer scenarios. Further analysis of SAPK dependent effectors as well as other components of stress signalling pathways will therefore be the focus of future research.
Figure 2. Inverse correlation of miR-335 expression and CHFR expression in the cisplatin-sensitive A2780 ovarian tumour cell line and the cisplatin-resistant CP70 cell line.
MiR-335 in therapeutic drug response
MicroRNAs (miRs) are important regulators of gene expression and their deregulation is a common feature in tumour cells. We identified miR-335 among microRNAs that are transcriptionally down-regulated upon expression of oncogenic Ras. Uniquely, miR-335 is up-regulated transcriptionally in cells after treatment with the DNA damaging agent cisplatin. Frequently, in ovarian tumour cells miR-335 expression is epigenetically silenced by methylation that can be reversed by treatment with demethylating drugs. In a cisplatin resistant derivative (CP70) of the ovarian tumour cell line A2780 expression of miR-335 significantly re-sensitises to cisplatin-induced apoptosis. This correlates with the finding of epigenetic silencing of the endogenous miR-335 gene locus in CP70 cells. In contrast, a putative miR-335 target gene, CHFR, is epigenetically silenced in A2780 cells but shows significant expression in CP70 cells. Thus, CHFR expression in A2780 cells may be suppressed by both epigenetic silencing through methylation and post-transcriptionally by miR-335. Suppression of CHFR in CP70 cells by siRNA leads to significant sensitisation to cisplatin. CHFR is a known modulator of chromatin modifying enzymes and we find that both expression of miR-335 and suppression of CHFR leads to altered expression of histone deacetylase, HDAC1. Our data therefore suggest that CHFR is a critical target for miR-335 mediated sensitisation to cisplatin.