Generation and side by side analysis of MMTV-Myc versus MMTV-Ras mice strains highlight a unique role for Gadd45a as either a suppressor or promoter of breast cancer development, employing distinct signaling pathways in response to distinct oncogenic stress stimuli ( and Supplemental Figure 7
). Our data indicates that the Gadd45a tumor suppressor function, mediated through activation of JNK and p38 stress kinases, contributes to Ras-induced apoptosis and senescence respectively, and is a unique response to Ras oncogenic stress. In contrast, the tumor promoter function of Gadd45a, mediated through negative regulation of MMP10 expression via the GSK3β/β-catenin signaling cascade, resulting in increased tumor vascularization, is a unique response to oncogenic Myc. These novel results indicate that Gadd45a can function to either promote or suppress breast tumor development through engagement of different signaling pathways, depending on the molecular nature of the activated oncogene.
Schematic diagram demonstrating how gadd45a modulates mammary tumor development in Myc-driven tumors compared to Ras-driven tumors
In the case of Myc-driven breast carcinogenesis, we have shown that the loss of Gadd45a results in increased levels of MMP10 through inactivation of the GSK3β/β-catenin signaling pathway (). MMP10 was identified as a direct target of β-catenin. Loss of Gadd45a increased phosphorylation of the GSK3-β kinase, rendering it inactive, and dramatically increased the levels of β-catenin. Inactivation of GSK3-β by phosphorylation is known to result in accumulation of hypo-phosphorylated β-catenin, which in turn translocates to the nucleus where it binds to TCF and functions as a transcriptional activator24
. It is noteworthy that while our work was in progress evidence was obtained that in keratinocytes, UV induced Gadd45a directly associates with GSK3β to promote GSK3β dephosphorylation and activation26
. These findings are supported by our data providing direct evidence that in normal mammary tissue, Gadd45a promotes dephosphorylation of GSK3-β
MMP10 is known to play a role in capillary tubular network collapse and regression due to its ability to degrade various components of basement membrane matrix, such as collagen type IV, laminin and proteoglycans21
. Thus, it is logical to assume that the decrease in vascularization that is observed in Myc expressing tumors deficient for Gadd45a is a direct consequence of MMP10 activation. In turn, the massive levels of cellular death, associated with accumulation of cellular debris and increased apoptosis observed in Myc expressing tumors deficient for Gadd45, is likely to be the consequence of the decrease in vascularization. It is notable that the massive cell death, as evident by large areas of cellular debris and nuclei fragments, observed upon histological analysis of Myc/Gadd45a−/− tumor sections (), by far exceeds the percentage of apoptotic cell death detected by the TUNEL assay (). Keeping in mind that the TUNEL immunohistochemical data in essence is a snapshot of cells undergoing apoptosis at the time of tumor fixation, the histological data reflects accumulative apoptotic cell death during tumor development. However, it is also possible that in addition to apoptosis, necrosis contributes to Myc/Gadd45a−/− tumor cell death. The increase in cell senescence in Myc expressing tumor tissue deficient for Gadd45a may also be due at least partially to the decrease in vascularization. Alternatively or in addition, it may also be the consequence of the role implicated for MMP10 as a regulator of cellular senescence27
. Whatever may be the case, the in vivo RNAi mediated suppression of MMP10 expression correlating with increases in Myc-driven tumor growth, provides direct evidence for a novel role of MMP10 in suppression of breast carcinogenesis.
The notion that the response of Gadd45a to oncogenic stress signals depends on the nature of the activated oncogenes is novel and intriguing. These results stress the significance of the type of oncogenic alterations found in the target cell on how stress response genes, such as Gadd45a, influence the initiation and progression of tumors. Since Gadd45a is a transcriptional target for both BRCA1 and p53, our observations regarding the unique role played by Gadd45a as either a tumor suppressor or tumor promoter, depending on the activated oncogene, also has important implications regarding the role of BRCA1 and p53 as tumor suppressors in the context of distinct oncogenic stressors. It is noteworthy that deficiency of the CDKI p21 was observed to differentially affect Ras- versus Myc-driven mammary tumor properties, promoting either growth arrest or proliferation, depending on the specific cellular context, although an exact mechanism was not identified28
Finally, we would like to point out that our findings raise several interesting questions, which warrant further investigation: (1) How does Gadd45a interface with different signaling cascades in response to distinct oncogenic stress; (2) How does Gadd45a influence breast carcinogenesis driven by oncogenes other than Ras and Myc; (3) How does Gadd45a modulate development of other tumor types driven by distinct oncogenic stress; (4) What role does Gadd45a expression or the lack of it play in human breast carcinogenicity and finally, (5) Do other Gadd45 proteins, i.e. Gadd45b and Gadd45g, either separately or in combination with Gadd45a, modulate breast tumor development. Current research is targeted at addressing these interesting issues.