The Notch pathway

Notch gene mutations were first discovered in Drosophila via malformations of the wing [69]. This ligand-activated signaling pathway is a highly conserved mechanism for maintaining stem cell function and regulating apoptosis, proliferation and cell fate specification [69]. Mammals express four Notch receptor family members, termed Notch 1-4 and five ligands; two Jagged family ligands (jagged-1 and jagged-2) and three delta-like li-gands (Dll1, Dll3 and Dll4) [69]. The Notch receptors are highly similar in structure and the extracellular domains contain epidermal growth factor-like repeats. The Notch li-

gands are also transmembrane proteins. Thus, the Notch receptors regulate cell behavior via juxatacrine signaling that requires direct contact between the ligand-expressing cells and those cells expressing the receptor. Ligand binding activates two consecutive proteolytic cleavages to free the intracellular portion of the receptor, which is referred to as the Notch intracellular domain (NICD) [70]. The first cleavage is carried out by an A Disintegrin And Metalloprotease (ADAM)-family of transmembrane metalloproteases. The second cleavage is carried out by y-secretase, an integral membrane enzyme complex, that is perhaps best known for its role in generating the amyloid-beta peptide found in brains of Alzheimer's disease patients [70, 71]. The NICD is a transcriptional co-activator. Once released, it travels to the nucleus via a nuclear transport signal where it binds to DNA-bound CSL. NICD binding to CSL displaces repressor complexes and recruits the mastermind family (MAML, mastermind like) of transcriptional coactivators, thereby activating the transcription of Notch-responsive genes [69]. In the absence of Notch receptor activation, CSL nucleates transcriptional repressive complexes via recruitment of histone deacetylase activities through interaction with SHARP (SMART and HDAC associated repressor) and corepressors like SMART/NcoR, CtIP/CtBP or ETO family members [72]. Interestingly, ETO (also called MTG8) is the target of the t(8;21) that produces a RUNX1/ETO fusion gene. Thus, the t(8;21) targets components of both the CBF and Notch pathways, highlighting yet another way in which these pathways intersect.

To date, a limited number of Notch-responsive genes have been identified. Some of the first gene targets identified include the transcription factors Hairy and enhancer of split-1 (Hes1) and Hairy and enhancer-of-split related with YRPW motif 1 (Hey1). Both Hes1 and Hey1 can be activated by a constitutively activated Notch1 receptor suggesting that these genes are bona fide targets [69]. Other CSL target genes are important mediators of signaling, including Akt and NF-kB, and important cell cycle regulators such as c-myc, D-type cyclins, p21Wafi/ciPi and p53 [69]. CSL is the only down-stream transcription factor directly responsive to Notch activation and, therefore, is crucial to Notch function.

The Notch pathway is deregulated in a variety of leukemias and solid cancers. For example, the mammalian orthologue of Notch was identified as TAN1 the target of the t(7;9) (q34;q34.4) in T-cell acute lymphocytic leukemia (T-ALL). While the t(7;9) is relatively rare (1% of all T-ALL) [73], the Notch1 receptor is constitutively activated by point mutations in the majority of T-ALL (almost 60%) [74]. Subsequent to the identification of Notch alterations in T-cell leukemia, the Notch pathway has been implicated in a variety of other human malignancies including cancers of the breast, ovarian, prostate, colorectal, and pancreas, as well as other leukemias [75-78]. In breast cancer, the Notch pathway components are commonly over-expressed and increased expression of Notch or Jag1 correlates with poor prognosis [76]. More recently, some studies suggest that breast cancer stem cell fate is regulated through the Notch pathway [79]. The Notch pathway is required for normal development of the murine prostate, and like breast cancers, prostate cancers also utilize the Notch pathway [80]. For example, Notch-1 and Jagged-1 expression constitute part of a gene expression signature for prostate cancer [81]. Other evidence indicating a role for Notch signaling in pros tate cancer includes studies showing that Jagged-1 expression correlates with prostate cancer recurrence and proliferation of prostate cancer cell lines [82, 83]. Moreover, down-regulation of both Notch-1 and Jagged-1 expression in the androgen insensitive prostate cancer cell line, PC3, was associated with a loss of malignancy and a reduction in Akt, mTOR and NF-kB activation [84].

As discussed above, the constitutive activation of Notch receptor signaling in diverse cancers is well documented, but the contribution of CSL to Notch-dependent oncogenesis has not been well studied. Our recent publication was the first to demonstrate that CSL was essential for the growth of prostate and breast cancer derived cell lines [85]. In these cancer cells, where Notch signaling is constitutive, CSL is required for growth in vitro. Thus, CSL is not only the focal point of Notch-dependent transcriptional control but appears to be central to the oncogenic Notch pathway as well [85].

In addition to the oncogenic functions associated with Notch signaling, the Notch pathway can also be tumor suppressive in cells or tissues where Notch predominately promotes differentiation [86]. Notch associated tumor suppressor activity is best illustrated in carcinoma of the skin, where keratinocyte specific inactivation of Notch1, Delta-like 1 (Dl1) or y-secre-tase treatment accentuates tumor formation in chemical carcinogenesis models [87]. Increasingly, tumor suppressive activities of the Notch pathway are being reported, as interest in Notch signaling and the use of y-secretase inhibitors to block Notch receptor activation has expanded. Inactivating mutations of Notch1 have been identified in head and neck squamous cell carcinoma [88] and haploinsufficiency of Notch1 or inhibition of Notch signaling with monoclonal antibodies to the Notch ligand Delta-like 4 induces vascular tumors in model systems [89]. As if to highlight the context dependent nature of Notch signaling, one report provided evidence that activated Notch1 alleles cooperated with oncogenic Ras to induce pancreatic cancer while a second report indicated that inactivation of Notch1 cooperated with Ras pathways in pancreatic cancers [90, 91]. This duality of function associated with Notch signaling has led to serious concerns regarding Notch receptor activation as a target of therapeutic intervention [86].

In prostate cancer, like in other cancers discussed above, Notch pathway signaling can be tumor suppressive. For example, NICD activity and Hes1 expression have been observed to be high in benign prostatic hyperplasia but low in prostate cancer indicating that Notch pathway activation can be lost during malignant transformation. Additionally, activation of the Notch pathway in the androgen independent prostate cancer cell line, DU145, inhibited cell growth and resulted in the activation of the PTEN tumor suppressor. Interestingly, knockdown of CSL in the DU145 cell line results in loss of cell growth (Yong and Davis, unpublished data). These data demonstrate that CSL (in a repressed complex) is required in cells where the Notch pathway can display tumor-suppressing activity. Clearly, the activity of the Notch pathway in prostate cancer is context dependent and complex.

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