Membranous E-cadherin expression was positive in all 57 cases (100%), whereas cytoplasmic E-cadherin staining was more prevalent in the more aggressive B and C groups with 21%(5/24) and 7% (1/14) cytoplasmic positive staining, respectively. Of all group A cases (n = 19), none (0%) were negative for vimentin and EGFR, 15 (79%) were positive for PDGF-D, and 6 (32%) were positive for nuclear NF-κB (; ). In all group B cases (n = 24), 18 (75%) were positive for PDGF-D, 10 (42%) were positive for NF-κB, 6 (25%) were positive for vimentin, and 2 (8%) were positive for EGFR. In group C cases (triple-negative; n = 14), there was increased expression of vimentin in 9 cases (64%), of EGFR in 14 cases (100%), of NF-κB in 11 cases (79%), and of PDGF-D in 9 cases (64%). We found increased expression of vimentin (P < .0002), EGFR (P < .0001), and NF-κB in triple-negative breast cancer specimens, which was statistically highly significant (P < .02) when compared with groups A and B.
Figure 1 Microphotographs of representative examples of immunohistochemical staining of EMT markers in breast cancer cases showing (A) vimentin cytoplasmic positivity in group C, (B) EGFR membranous and cytoplasmic positivity in group C, (C) NF-κB nuclear (more ...)
EMT Markers in Breast Cancer Cases.
Breast cancer is a heterogeneous disease that consists of multiple molecular subtypes. The presence of hormone receptors ER and PR and overexpression of human EGFR-2 (HER-2) is of central importance in the therapeutic decision-making process for patients with breast cancer. Apart from predicting response to therapy, these factors may also determine the likelihood of disease relapse. Hormone receptor-positive tumors have been considered to have favorable outcome because of their response to endocrine manipulations such as tamoxifen, aromatase inhibitors, or ovarian ablation. Triple-negative breast cancer (TNBC) is a term that has been used to describe a biologically diverse group of breast tumors that are lacking in the expression of ER, PR, and HER-2. Tumors with a triple-negative phenotype tend to have poor prognosis and, unlike their hormone receptor or HER-2-positive counterparts, lack targeted therapeutics. As a result, the interest in this aggressive TNBC from both clinicians and scientists has grown exponentially.
The EMT phenomenon is triggered by the interplay of several extracellular signals; many secreted soluble factors, growth factors, their effectors, and many transcription factors including PDGF, Notch, and NF-κB [6
]. The translational relevance of these EMT markers in evaluating aggressiveness of human breast cancers specimens has not been well investigated, and thus in the present study, we interrogated EMT markers such as E-cadherin, vimentin, NF-κB, EGFR, and PDGF-D in breast cancer specimens. Cases were divided based on their prognosis and aggressiveness into the highly aggressive triple-negative tumors versus
the relatively innocuous ER-, PR-, HER-2-positive tumors. EMT markers (vimentin, EGFR, and NF-κB) were found to be significantly higher in triple-negative breast tumors (known aggressive tumors) but not PDGF-D, and our findings are consistent with similar findings reported in the literature [12
Vimentin is a cytoplasmic intermediate filament protein found to be the major component of the cytoskeleton. Our findings of its association with clinically aggressive behavior of breast tumors are also consistent with previous studies [13–15
]. Conversely, Heatley et al. [16
] have demonstrated that vimentin expression did not inversely predict patient survival. The association of vimentin with clinically aggressive behavior of tumors has been explained based on the correlation of vimentin's expression with lack of steroid receptors and poor differentiation of cancer [9
]. During EMT, the cell intermediate filament status changes from a keratin-rich network, which connects to adherens junctions and hemidesmosomes, to a vimentin-rich network connecting to focal adhesions [17
]. Sarrio et al. [18
] have suggested that vimentin-positive cells may have a phenotypic plasticity prone to undergo EMT, and although transient, they involve modulation of a number of EMT genes.
The EGFR is a member of the ErbB family of receptor tyrosine kinases. The high incidence of EGFR expression in triple-negative tumors in our study is similar to findings by other authors [19,20
]. The EGFR positivity has been related to a less favorable response to chemotherapy and poorer survival, indicating that it can serve as a valuable tool for selecting appropriate treatment regimens for patients with TNBC [19
]. The activation of EGFR leads to the activation of NF-κB, which could be useful for predicting tumor aggressiveness. NF-κB is a transcription factor that is well known to contribute to the acquisition of EMT and tumor cell invasion [3
]. High NF-κB expression found in the triple-negative breast cancer cases in our study is similar to those of previous authors [21,22
]. Genes induced by the NF-κB activation could serve as therapeutic targets of triple-negative breast cancers, and to that end, inactivation of NF-κB pathway by many natural agents has been reported [23–26
], including recent findings of plumbagin, suggesting that natural agents could be useful for the prevention and/or treatment of aggressive breast cancer especially the TNBCs [27
PDGF-D is an important regulator of cell proliferation, transformation, invasion, and metastasis in human cancer [10
]. It has been linked with several human malignancies [28
] and has also been shown to play an important role in the processes of EMT [29,30
] by causing changes in cellular morphology concomitant with loss of E-cadherin, gain of vimentin, increase in tumor growth, and increased cancer cell invasion and angiogenesis [10
]. Moreover, forced overexpression of PDGF-D in PC prostate cancer cells showed the acquisition of stem cell characteristics, suggesting the role of PDGF-D in self-renewal and tumor cell aggressiveness [31
]. In the present study, we found that PDGF-D was not significantly higher in triple-negative breast tumors when compared with the other prognostic groups. A review of literature reveals that similar studies have not been previously done in human breast cancer tissues; however, in breast cancer cell lines, PDGF-D has been demonstrated to play an important role in tumor aggressiveness [28
]. Several reasons might be behind the lack of correlation between tumor aggressiveness and PDGF-D positivity in our study. First, the use of IHC to evaluate protein levels does not always reflect the structure or functionality of the protein. Second, the small size of the patient cohort would introduce an element of bias in this evaluation. Third, technical aspects such as the clone of antibody used and antigen retrieval methods might have played a part in this disparity and needs further clarification in future IHC studies.
In summary, based on our findings, we conclude that the expression of vimentin, EGFR, and NF-κB were significantly increased in human TNBCs. The higher expression of these markers seen in triplenegative tumors may explain the different biologic behavior of these tumor types. Therefore, vimentin, EGFR, and NF-κB may have the potential to be used as markers for EMT in breast cancers, and targeted inactivation of these markers could be useful for designing personalized medicine, which would be the future to make an impact on improving the overall survival of patients diagnosed with TNBCs.