Unlike T-cell acute lymphoblastic leukemia (T-ALL), in which more than half of patients have an activating mutation in Notch1 [8
], a chromosomal translocation involving Notch loci or an activating mutation has yet to be reported in breast cancer. In T-ALL, activating mutations are commonly found in the Notch1 NRR that regulates NICD production and in the proline/glutamic acid/serine/threonine-rich (PEST) domain, which regulates NICD turnover. However, among 48 breast cancer samples, only one nonsense mutation in Notch2, which produced a truncated PEST domain and potentially enhanced Notch2 signaling, was found [9
]. No mutations in the other three Notch receptors were observed. However, as will be discussed later, the functional consequence of Notch2 activation - suppressing or promoting breast tumorigenesis - remains to be investigated. The absence of mutations raises the critical question of how Notch signaling could be activated and pathological in breast cancer. Two possible explanations have been proposed: (a) higher expression of Notch receptors or ligands or both and (b) loss of the negative regulator, Numb.
Several studies reported that the expression of Notch receptor and ligand proteins is higher in breast cancer tissues than in normal breast epithelium [10
]. Rizzo and colleagues [12
] examined the expression of Notch1, Notch4, Jagged1, and Dll1 in 4 normal breast tissues, 5 hyperplasias of usual type (HUTs), 27 ductal carcinomas in situ
(DCISs), 27 infiltrating ductal carcinomas (IDCs), and 14 infiltrating lobular carcinomas (ILCs) by immunohistochemistry. None of the normal breast tissues expressed high levels of Notch1 or Notch4. In contrast, 80% of HUTs, 67% of DCISs, 89% of IDCs, and 57% of ILCs expressed high levels of Notch1. High Notch4 levels were not detected in any HUT or DCIS samples but were present in 81% of IDCs and 93% of ILCs. The expression of Jagged1 and Dll1 was not examined in normal tissues, HUTs, or DCISs, but 78% of IDCs and 64% of ILCs expressed high Jagged1 levels. Similarly, another study reported higher expression level of multiple Notch receptors and ligands in IDC compared with normal breast tissues [13
In a later study, Zardawi and colleagues [11
] used tissue microarrays to examine Notch1 expression in 693 samples from 222 patients. Surprisingly, although the authors found a gradual increase in the percentage of high Notch1 expression cases from normal breast tissues (13.6%) to grade 2 or 3 DCISs (57% to 59%), which is consistent with the notion that Notch1 signaling is activated in breast cancer and promotes tumor progression, the expression level of Notch in IDCs was lower than that in DCISs. More surprisingly, a recent study that examined 79 normal breast and 408 breast cancer samples, including 367 IDC and 29 ILC cases, found no difference in the expression of Notch1 protein between breast cancer and normal breast tissues [14
]. Therefore, whether expression of Notch receptors and ligands, especially Notch1 protein, is elevated in breast cancer tissues, or whether their expression level is increased during early stages of breast cancer development but declines during later stages, remains to be clarified.
Another proposed mechanism for aberrant Notch activation in breast cancer is the loss of a Notch-negative regulator, Numb. It is generally believed that Numb downregulates Notch signaling by promoting its polyubiquitination and lysosomal degradation or by regulating its endocytosis (or both), consequently reducing its availability on the cell surface, where it responds to ligand binding-induced activation [15
]. Numb was found to be absent or at low levels in approximately 50% of breast cancer samples and this was due to enhanced proteasomal degradation [16
]. Consistent with this, Stylianou and colleagues [17
] detected N1ICD (the active form of Notch1) in 20 breast cancer samples from which Numb was absent. However, the correlation between Notch activation and loss of Numb needs to be examined in a larger group of patients as a recent study of 36 IDC cases failed to demonstrate a direct correlation [13
It should be emphasized that, while both elevated expression of Notch receptors and ligands and loss of Numb suggest the possibility of aberrant Notch activation, they are not, per se, equivalent to enhanced Notch signaling, which needs to be verified by the presence of active NICD and upregulated expression of Notch target genes. To this end, Mittal and colleagues [13
] reported that both active N1ICD and Hes1/5 protein could be detected in 27 out of 35 IDC samples but were present in only 1 of 13 normal breast tissues. N1ICD was detected in the nucleus and cytoplasm. Since active NICD in cultured breast cancer cells is always present in nuclei, the nature of cytoplasmic N1ICD in this study needs to be clarified further, as does the specificity of the primary antibody used. Furthermore, others have failed to find a difference in Hes1 expression when comparing normal breast tissues with breast cancer tissues [18
]. This raises concerns as to whether Notch is aberrantly activated or whether Hes1 is even a Notch target gene in breast cancer tissues.
Even if Notch signaling is more active in breast cancer compared with normal breast tissue, it remains to be determined whether the activation is sufficient to drive breast oncogenesis. Also, it is not known whether breast tumorigenesis requires the cooperation of other signaling pathways with Notch. This might be expected given that several activating Notch mutant alleles commonly found in T-ALL cannot initiate oncogenesis alone, despite their ability to activate several Notch target genes [19