Notch signaling pathway is known to play a vital role in normal development and growth. Notch signaling pathway is involved in embryonic development, cellular differentiation, proliferation, homeostasis and apoptosis. Numerous studies have reported that aberrant Notch expression is involved in neoplastic transformation and tumorigenesis. There are four known Notch paralogs which display different patterns in their expression and function across different malignancies. Oncogenic role of Notch signaling was first reported in T-ALL [17] and later reported in lung cancer, ovarian cancer, breast cancer, colorectal cancer [18, 19, 20, 21, 22] while tumor suppressive role of Notch signaling was reported in pancreatic cancer and hepatocellular carcinoma [23, 24]. However, research about relationship between Notch receptors and TNBC, and the association among the Notch receptors is limited and unclear.
In this study, we have examined the role of Notch receptors in TNBC cohort by analyzing the cellular localization of all four known Notch receptors by immunohistochemistry. We observed that Notch1 was expressed in the nucleus of tumor cells suggesting the translocation and accumulation of Notch intracellular domain (NICD1) in the nucleus of tumor cells. The accumulation of NICD1 in the nucleus was strongly correlated with high BR score and necrosis, indicating the tumor aggravating role of Notch1. Our data was in concordance with studies reporting the association of high grade tumors and poor prognosis of TNBC patients with up-regulated Notch1 [25, 26, 27].
Furthermore, upon examination of Notch2 protein it was revealed that Notch2 was localized in the cytoplasm and nucleus of tumor cells. Despite, various studies showing evidence of tumor suppressive role of Notch2 [28, 29, 30], our data suggests that Notch2 receptor plays a tumor promoting role in TNBC. It was observed that high cytoplasmic Notch2 expression was significantly correlated with local or distant metastasis, however nuclear and cytonuclear expression of Notch2 did not show any statistical significance with clinicopathological parameters. Nonetheless, our study was in line with evidence presented by Lee et al. and Sehrawat et al., stating that inhibition of Notch2 was responsible for anti-apoptotic activity and inhibition of migration in TNBC cells [31, 32]. Also, our study demonstrated that high levels of cytoplasmic Notch2 was positively correlated with worse disease-free survival and poor overall survival proving its tumor promoting role. Earlier studies regarding Notch pathway emphasized that nuclear translocation of NICD was required in order to trigger the downstream target genes. Although, recently it has been demonstrated that in non-canonical Notch pathway, nuclear translocation is not necessary for the pathway to be activated and it may interact with other molecules in the cytoplasm to activate the pathway [33]. Therefore, it may be suggested that the association between cytoplasmic Notch2 expression and poor prognosis of the patients might be due to non-canonical Notch pathway.
Moreover, our data represents that majority of TNBC samples exhibited high Notch3 protein levels in the cytoplasm. Along with cytoplasmic expression, membrane, cytomembrane and cytonuclear protein expression were also detected. Notably, it was revealed that up-regulated cytoplasmic Notch3 expression in tumors was associated with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status indicating that patients with up-regulated cytoplasmic Notch3 might have favorable prognosis. Our data was in line with studies demonstrating tumor suppressive role of Notch3. Zhang and colleagues observed that high expression of Notch3 inhibited cellular proliferation and tumorigenesis by activating PTEN resulting in better prognosis of breast cancer patients [34]. Another study by Chen and colleagues provided evidence that overexpression of Notch3 inhibited cell cycle progression by upregulating levels of Cdh1 in MDA-MB-231 cell lines. However, contrary to above findings, it was observed that membrane and cytonuclear expression of Notch3 were strongly correlated with poorly differentiated tumors and necrosis respectively. Also, a trend of association was observed between frequent membranous Notch3 expression and metastasis. Although, cytonuclear expression of Notch3 was not detected frequently it was strongly associated with necrotic tumors suggesting the role of nuclear translocation in tumor promoting activity of Notch3. These results were parallel to the findings of Choy and colleagues that revealed the involvement of nuclear Notch3 expression in tumor promotion [35]. The association of increased levels of Notch3 with poorly differentiated tumors might be due to the non-canonical pathway, where membrane-bound Notch3 may have interacted with cell regulators present in the cytoplasm. Contrary to that, membrane Notch3 expression was tended to associate with low BR score tumors. These results of Notch3 suggests that nuclear translocation of Notch3 is potentially involved in promoting tumor aggravating activity, whereas membrane-bound Notch3 may act as an oncogene via activation of non-canonical pathway which does not essentially require nuclear translocation. However, the strong association of high cytoplasmic Notch3 expression with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status might be due the immature state of the receptor or interaction of cytoplasmic Notch3 with tumor suppressing molecules which might be responsible for favorable prognosis of TNBC patients.
In addition, in the majority of the patients expressing Notch4 receptor, it was frequently detected in the cytoplasm. This study revealed that high levels of cytoplasmic Notch4 were negatively associated with younger age and necrotic tumors. Contrary, to a study by Wang et al. which stated that Notch4 was associated with poor prognosis for TNBC patients [36], our data represents that Notch4 is inversely correlated to poor prognostic factors of the disease and may function as a tumor suppressor. Concurrently, few studies have presented over expression of Notch4 receptor as tumor suppressive in melanoma and endometrial cancer [37, 38].
Further, we evaluated the co-expression patterns of Notch receptors in TNBC patients. It was observed that Notch2 and Notch3 receptors showed positive correlation with Notch4 receptor. This may be interpreted as Notch receptors are significantly associated with each other and even though Notch4 might function as a good prognostic factor, together with Notch2 and Notch3 receptors they contribute to tumor promotion and poor survival of TNBC patients. Moreover, it was observed that nuclear expression of Notch3 was positively associated with nuclear expression of Notch1 suggesting the oncogenic role of nuclear localization of both Notch1 and Notch3 receptors. Additionally, it was noted that nuclear Notch1 expression was negatively correlated with the cytoplasmic Notch3 expression indicating opposing role of both the receptors based on their localization, as cytoplasmic Notch3 is associated with better prognosis and nuclear Notch1 is linked to poor outcome of the disease. Therefore, our results imply that interplay of Notch receptors is rather complex mechanism.
In conclusion, our study demonstrated that Notch receptors exhibit a pleiotropic role in TNBC. Notch1 and Notch2 receptors play a tumor promoting role and contribute to poor outcome in TNBC patients. It was also revealed that although membrane and cytonuclear Notch3 had tumor aggravating effects, cytoplasmic Notch3 and Notch4 receptor had tumor suppressive effect. It is implicated that the tumor promoting activity of Notch receptors might be due to the canonical Notch signaling pathway which essentially requires the nuclear translocation of NICD to trigger the transcriptional regulation of target genes or it might be due to the non-canonical pathway which is activated through the interaction of Notch receptors with transcriptional regulators present in the cytoplasm to exert its effects. The tumor suppressive effect of cytoplasmic Notch3 and Notch4 expression may be due to the inactive state of the receptor or its interaction with tumor suppressive molecules in the cytoplasm. Therefore, it is important to assess the subcellular localization of the Notch receptors as they are ubiquitously distributed in the cells in order to evaluate their individual effects in TNBC. Hence, our results strongly indicate that Notch receptors are involved in promoting TNBC and chiefly, Notch2 receptor may serve as a potential prognostic marker and therapeutic target for patients with TNBC.