This study examined the expression of EGFR and its activated form, pEGFR, in invasive breast carcinoma. We demonstrated that EGFR and pEGFR are both expressed in the membrane of 11.3% and 35.7% of tumour cells. The greater rate of positivity for pEGFR than for EGFR could be attributed to the EGFR downregulation process. Once EGFR is activated it undergoes internalization, resulting in a marked decrease in nonactivated membrane-bound EGFR [24
]. In previous studies, EGFR immunopositivity exhibits great variety [7
]. The wide range of EGFR expression (14% to 91%) may be accounted for by use of different methods and different criteria for assessment, as well by the presence of basal-like carcinomas that consistently over-express EGFR.
None of the studied proteins was associated with menopausal status, tumor size, lymph node status, tumour stage and PR hormonal status, which is in accordance with the majority of reports on EGFR expression [9
]. The absence of any association between EGFR and c-erbB-2 expression may be due to variations in c-erbB-4 expression, which antagonizes the influence of c-erbB-2 in tumors [7
With regard to EGFR, we showed that its expression was positively associated with nuclear grade (P
= 0.001) and inversely associated with ER hormonal status (P
= 0.005), which is in accordance with a large number of studies [25
]. This consistent finding in much of the literature had led to the plausible hypothesis that EGFR signalling may be associated with endocrine resistance or insensitivity [27
]. Alternatively, this finding may be accounted for by the strong association between EGFR expression and loss of differentiation in breast cancer.
EGFR activation has an antiapoptotic effect through PI3K pathway [1
], a fact that was corroborated by the pEGFR relationship to pAkt (P
= 0.014). EGFR can lead to activation of PI3K both directly and indirectly through Ras; it induces downstream activation of phosphoinositide-dependent protein kinase-1 and -2 that phosphorylate thr308 and ser473 on Akt, respectively. Akt's antiapoptotic role is well known, via phosphorylating and sequestering downstream targets including the FOXO family of forkhead transcription factors, the proapoptotic Bad and the protease caspase-9, and by activating the pro-survival transcriptional regulator protein nuclear factor-κB.
Expression of pEGFR was associated with VEGFR-1/Flt-1 and MMP-14. VEGFR-1/Flt-1 is a specific endothelial cell receptor to which the angiogenic factors VEGF-A and VEGF-B bind; it promotes differentiation and vascular maintenance [30
]. Indeed, in tumour progression EGFR upregulates VEGFR; thus, it is implicated in angiogenesis. In addition, in human cancer cells the EGFR autocrine pathway controls the production of several proangiogenic growth factors, including VEGF [4
]. It is also known that several EGFR inhibitors, such as monoclonal antibodies, result in a concurrent downregulation of tumour-induced, VEGF-mediated angiogenesis [4
]. Therefore, the above-mentioned relationship implies an angiogenetic role of activated EGFR. The angiogenetic ability of pEGFR has further confirmed by the pEGFR association with pAkt, which is known to modulate angiogenesis via activation of endothelial nitric oxide synthase [31
]. MMP-14, as well as most of the MMPs, may promote angiogenesis by at least two different mechanisms: by degrading barriers and allowing endothelial cell invasion; or by liberating factors that promote or maintain the angiogenic phenotype [32
]. In addition, it promotes invasion and metastasis by degrading ectracellular matrix [33
]. It appears that the above-mentioned relationship between EGFR and MMP-14 reflects the well established interaction between EGFR pathway and the MMPs [1
]. EGFR activation is able to upregulate MMPs, whereas MMPs participate in several pathways of EGFR activation, such as the ectodomain shedding of EGFR transmembrane precursor [4
], G protein-coupled receptor-mediated transactivation, and uPAR-mediated transactivation of EGFR [1
The combination of EGFR/pEGFR was associated with pAkt, MMP-14 and VEGFR-1, which may be accounted for by the parallel relationship between pEGFR and those biological parameters.
Another observation in the present study, that of the parallel relationship between pEGFR and uPAR, supports the existence of a uPAR-mediated EGFR transactivation pathway [1
] and enforces the invasive effect of pEGFR. The uPAR glycoprotein, receptor of plasminogen activation system, plays a central role in extracellular matrix degradation; thus, it participates in tumour invasion and metastasis [34
]. Previously, in breast cancer, it has been related to tumour aggressiveness and patients poor survival [21
With regard to the association of EGFR and pEGFR with prognosis and survival, we found that EGFR and its active form are significantly associated with overall survival (P
= 0.035 and P
= 0.016, respectively) but not with disease-free survival in univariate analysis. Findings from previous studies regarding prognostic significance of EGFR are controversial. Tsuitsu and coworkers [11
] reported the prognostic value of EGFR for overall and disease-free survival, whereas other investigators failed to confirm its prognostic significance in either the entirety of studied cases [7
] or in lymph node positive [8
] or -negative cases [35
]. On the other hand, there is no report about pEGFR expression and prognosis in breast cancer, as far as we know, whereas in our study pEGFR appeared to have an unfavourable impact on overall survival. We also observed that EGFR-positive/pEGFR-positive tumours had worse prognosis in contrast to EGFR-negative and/or pEGFR-negative ones. Moreover, EGFR/pEGFR was found to be an independent prognostic indicator for overall survival. EGFR and pEGFR coexpression appears to be more representative of EGFR dynamics in tumour than solely EGFR or pEGFR expression. Probably, in EGFR-positive/pEGFR-positive tumours, the total number of receptors is greater; thus, there are more receptors available for subsequent phosphorylation. Another possible explanation might be the existence of an inhibitory mechanism, which impedes autophosphorylation of several receptors and permits only a few receptors to become phosphorylated and initiate EGFR-dependent signalling cascades.
Nieto and coworkers [37
] also studied EGFR and pEGFR expression in invasive breast cancer, but they failed to attribute any prognostic value to pEGFR expression. To the best of our knowledge, there are no published data confirming the prognostic value of pEGFR or EGFR/pEGFR coexpression in breast cancer. Interestingly, Arteaga and Baselga [14
] have noticed that EGFR-positive colon carcinomas with simultaneous expression of pEGFR, coexpressed the EGFR ligand transforming growth factor-α and markers of tumour proliferation, which is in contrast to EGFR-positive/pEGFR-negative carcinomas. Thus, EGFR content in EGFR-positive/pEGFR-negative tumours does not reflect the level of receptor activation.