Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers

Several studies have emphasised the role of MAPKs in cancer progression [13, 29‐31]. The functions of MAPKs in BC appear to be complex owing to several cellular responses that they modulate and their interaction with different pathways including the key BC genes ER and HER2. In the current study, the role of MAPKs in BC and how the expression of ER and HER2 might influence their function were investigated using a large panel of MAPK proteins and the results were validated in vitro using RPPA and different BC cell lines. The results showed that generally most of MAPKs are associated with good prognostic features in the whole series and in the ER+ tumours. MAPKs are mainly related to ER expression and this finding was observed using IHC and validated by RPPA.

Our results are consistent with others [32] who demonstrated that ERK1/2 and p-ERK1/2 were associated with good clinicopathological variables and that by Hsu et al. [33] who found that p-ERK1/2 is required for inducing apoptosis especially in MCF-7 and MDA-231 cell lines. The association between MAPKs and good prognostic variable may be related to their roles in inducing apoptosis. p-JNK1/2 also has been shown to be stimulated by stress or growth factors and either one can enhance p-JNK1/2 to stimulate apoptosis [34] and even p-JNK1/2 augments cell death signalling in slowly growing MCF-7 cells under the influence of high estradiol level [35]. Interestingly, a drug “pseudolaric acid B” whose function mimics the upstream mediators of MAPKs was used in an in vitro experiment and this revealed the apoptotic function of JNK and its phosphorylated form upon activation [36]. Moreover, several studies have demonstrated the role of p-p38/MAPK in inducing apoptosis in BC and some suggested that this effect is mediated by TGF-β [37‐40]. p-ATF2 is thought to have dual functions independent of each other; the first is a tumour suppressor protein and the other function is related to DNA damage response pathway [41] and importantly, the former function has been confirmed by different studies [38, 42, 43]. The above results regarding the association between MAPKs and apoptosis were supported by our findings that MAPKs were positively associated with ER, BCL2 and negatively with HER2 (or no association with some of them), KI67-LI and p53.

Consistent with previous studies that assessed the prognostic value of individual MAPK members [32, 43, 44], in this study we identified an association between MAPKs proteins and better outcome in terms of longer survival time. Importantly, in the current research, pan ERK1/2 and p-ERK1/2 showed an association with better outcome in patients with ER+ tumours who are candidate for endocrine therapy. Consistent with this, Busch et al. [45] have demonstrated that high p-ERK1/2 was an independent predictor of better outcome in tamoxifen-treated patients. Thus, this study implies that MAPKs could be prospective surrogate biomarkers in assessing benefit from endocrine therapy.

In the current study, RPPA results were consistent with IHC findings for those proteins used in both techniques and the findings were also consistent for those MAPK members that were used only in RPPA. In the ER−/HER2+ cell line and in line with our results, a previous study demonstrated that activation of MAPK is associated with loss of ER phenotype especially in those overexpressing HER2 [46]. Ostrakhovitch et al. [47] have found that in MDA-231 cell line where p53 is mutated or suppressed, the phosphorylation of ERK1/2 was strong. Meanwhile, p-p38 overexpression in ER- tumours is thought to be associated with proliferation and progression of cancer. This is attributed to the fact that p-p38 can mediate proliferation only in BC cells that express mutant p53 and not the wild one, a finding commonly seen in ER− tumours rather than ER+ tumours [48]. Interestingly, Creighton et al. [49] indicated that hyperactivation of MAPK leads to the loss of ER expression and plays a role in the generation of the ER-negative phenotype. Although our study supports the differential expression of MAPKs between ER+ and ER− phenotypes, it indicates that MAPKs play an important role in ER+ tumours and that their expression in ER+ tumours is associated with better outcome. Supporting this, Atanaskova et al. [50] have indicated that activation of ER by MAPK enhances the expression of ER-regulated genes, accelerates tumour growth, and that MAPK/ER crosstalk enhances ER−mediated signalling without diminishing sensitivity to the inhibitory effects of anti-estrogens. However, in the ER-negative subgroup, it has been reported that the oncogenic effect of MAPKs is related to HER2 overexpression [49], which further support our findings.

The role of MAPKs in enhancing cell cycle arrest is also encountered. This has been attributed to the fact that innate tumour suppressor mechanisms are activated in response to aberrant oncogenic stimulation and remarkably induce growth inhibition that is referred to as oncogenic-induced senescence [51, 52]. In addition, accumulating evidence has demonstrated that sequestration of cytoplasmic ERK in the cytoplasm induced by proapoptotic molecules such as death-associated protein kinase could ultimately augment the apoptotic action of these proapoptotic proteins [53]. In this regard, a study revealed that feedback inhibitory signals are common from mutated RAS and RAF to the upstream mediators, which will omit further stimulation through this pathway being thoughtfully mediated by HDM2 and FOXO3 [54]. The previous findings might explain why high expression of MAPKs in ER+ tumours was associated with good features and outcome in our IHC findings and why the function is different in ER + and ER− cell lines by using RPPA. In addition, another reason which can explain the dual behaviour of these MAPKs is that they function in a cell context-specific and cell type-specific way to mediate signals that can lead to diverse cellular functions. Furthermore, the function of these MAPKs is influenced by their crosstalk and interaction with other pathways which can influence their behaviour [9‐11].

Despite the limitations of this study and type of techniques employed, the findings of this study collectively together with other previous publications mentioned above support the tumour suppressor role of MAPK kinase pathway in BC and that active pathway is associated with variables of good prognosis and better outcome. In addition, our results provide further evidence that ER is the main player related to the function of MAPK in BC; however, when HER2 is overexpressed, the role of ER becomes less significant and there is some evidence that HER2 can play a major role in controlling the MAPK pathway activation in ER-negative tumours.

In conclusion, our study illustrated the role of MAPKs and their signalling in BC and demonstrated that MAPKs are associated with good prognostic features and show differential expression within ER+ and ER− groups.