Notch and its oncogenic activity in human malignancies

It is generally accepted that Notch signaling plays a fundamental role during embryonic development that is associated with the control of cell proliferation, differentiation and apoptosis. Moreover, a great number of studies have revealed that this pathway also shows a connection to postnatal processes. Noteworthy among these are hematopoiesis, mammary gland development, maturation of gastrointestinal epithelium, immune regulation, angiogenesis and neural stem cell survival [1, 2].

The Notch signaling pathway is composed of Notch receptors and Notch ligands. In humans the Notch receptors include Notch14. All receptors and their ligands, e. g. delta-like ligand (DLL1–4) and Jagged (JAG1–2), belong to the family of the single-pass transmembrane proteins characterized by multiple epidermal growth factor (EGF)-like repeats in the extracellular region. Notch receptors are synthesized as inactive single peptide precursors. These form before reaching the plasma membrane and are proteolytically cleaved by a furin-like convertases in the trans-Golgi network. The first cleavage (S1) generates non-covalently bound heterodimers that are composed of the N-terminal ligand-accessible Notch extracellular domain (NECD), and a C-terminal Notch transmembrane fragment (NTM). This fragment is composed of the extracellular stub, transmembrane domain and Notch intracellular domain (NICD) (Fig. 1).

The significant feature of the Notch pathway is juxtacrine acting between neighboring cells. During the first step of activation, Notch ligands bind to Notch receptors on an adjacent cell. During the second step Notch receptors undergo conformational changes followed by the second cleavage (S2), catalyzed by a member of a disintegrin and metaloprotease (ADAM) family (ADAM17 or ADAM10). The third cleavage (S3) is made by a presenilin-dependent γ‑secretase protease complex (an integral membrane protein complex) consisting of presenilin 1 (PSEN1or PSEN2, nicastrin, presinilin enhancer 2 [PEN2]) and anterior pharynx-defective 1 (APH1). After that, the active NICD is released into the cytoplasm and then to the nucleus, where it binds to the ubiquitous transcription factor CSL (CBF1/supressor of hairless, and longevity-assurance gene-1). NICD is known to convert a large co-repressor complex into a transcriptional activating complex. This complex is primarily composed of NICD, CSL, mastermind-like protein (MAML; a transcriptional coactivator), SKIP (ski-interacting protein as a CBF1 binding protein) and p300, and in this form induces the transcription of Notch target genes. Noteworthy among these are genes encoding hairy enhancer of split (HES) family proteins, HES-related proteins (HEY), Notch-regulated ankyrin repeat protein and p21cip/waf1, cyclin D1 and 3, c‑myc and HER2 ([3, 4]; Fig. 1).

Increasing evidence has demonstrated that Notch signaling is deregulated in human hematological malignancies and solid tumors [5]. This signaling has a protumorigenic effect, but may also act as a tumor suppressor [6]. How induction of a single pathway gives rise to the opposite effects in different cell types is still unknown. The Notch pathway probably turns on or turns off different target genes and downstream pathways [7]. This paper discusses the oncogenic activity of Notch in human malignancies.

Numerous reports have indicated that alterations in the Notch pathway were originally associated with the pathogenesis of T‑cell acute lymphoblastic leukemia/lymphoma (T-ALL), an aggressive hematologic tumor affecting children and adolescents. Nevertheless, this disease also occurs in adults. Patients presenting with diffuse (>25%) bone marrow infiltration are diagnosed with T‑cell acute lymphoblastic leukemia, while those with mediastinal masses and limited bone marrow involvement are diagnosed as T‑cell acute lymphoblastic lymphoma [8].

The first evidence demonstrating that Notch might be the significant element in leukemogenesis came in the early 1990s when Ellisen and coworkers characterized a rare chromosomal translocation t(7;9)(q34;q34,3) involving the human Notch1 gene in T‑ALL [9]. However, the fundamental role of Notch1 in T‑ALL development was discovered in 2004 together with the discovery of activating Notch1 mutations in nearly 60% of TAALs. Weng et al. showed that Notch1 mutations occuring in the HD domain (20% of T‑ALL) resulted in a ligand-dependent induction of the receptor. In contrast, mutations in the PEST domain presenting in 15% of T‑ALLs were responsible for enhanced NICD1 stability and abnormally prolonged Notch1 activation [10]. Additionally, juxtamembrane expansion (JME) alleles and intragenic deletions encompassing the 5’ region of the Notch1 locus (Notch1 del-N) showed connections with overexpression of Notch1 in rare cases of T‑ALL [11, 12]. Several studies revealed that nearly 20% of T‑ALLs might be connected with mutations in the FBXW7 gene [13‐15]. Those mutations have been linked to Notch1 PEST mutations and they led to increased ICN1 stability. It should be pointed out that FBXW7 mutations may also be related to another oncogenic activity, since this F‑box protein is involved in the degradation of oncoproteins such as MYC, JUN, MCL1 and cyclin E [16, 17].

Recent studies demonstrated a close dependency between the protumorigenic effect of Notch1 and the transcriptional regulation of cell growth and metabolism. Notch1 directly controls a great number of genes involved in anabolic pathways and also promotes cell growth through direct transcriptional upregulation of MYC [18]. The major results of Notch1 activation during the regulation of cell growth are further supported by the interaction of Notch1 signaling with the PI3K-AKT-mTOR pathway [19‐21]. Notch1 transcriptionally upregulates molecules upstream of the PI3K such as the interleukin 7 receptor alpha chain (IL7RA), the pre-T-cell receptor alpha (PTCRA) and the insulin-like growth factor receptor (IGF1R) [22‐24].

Among other significant mechanisms associated with T‑cell transformation downstream of oncogenic Notch, mention should be made of the promotion of the G1-S phase. Notch1 signaling is associated with the promotion of G1-S through upregulation of CDK4 and CDK6 and downregulation of p27/KIP1 and p18/INK4C cell cycle inhibitors [25‐27]. Ntziachristos et al. demonstrated that Notch1 antagonized the activity of the Polycomb repressive complex 2 (PRC2) connected with writing the H3K27 mark. It is also worth noting that mutational loss of the PRC2 complex including EZH2, SUZI2, and EED has often been found in T‑ALL [28].

The question arises regarding the possible relation of Notch1 activation to clinical outcome in T‑ALL. Breit et al. showed that the presence of Notch1 mutations has been significantly correlated with prednisone response, favorable minimal residual disease (MRD) kinetics and improved long-term outcome in children with T‑ALL [29]. Analysis of the prognostic impact of both Notch1 and FBXW7 mutations in pediatric T‑ALL patients showed a favorable prognosis [30]. In contrast, Mansour et al. did not show any significant association between Notch1 and FBXw7 mutations and prognosis [31]. In this context, it is worth noting the study conducted by Ben Abdelali et al., who revealed that the favorable prognosis of Notch1/FBXW mutations in adult T‑ALL was found in more intense, pediatric-inspired GRAALL chemotherapy protocols but not in the less-intense LALA-94 study [32]. This could suggest that differences in therapy intensification may influence the prognostic impact of Notch activating mutations in T‑ALL.

It is worth noting here the identification of a novel chalcone derivative inhibiting Notch signaling in T‑ALL. By functional and biological analysis of the most representative molecules of an in-house library of natural products, Mori et al. designed and synthesized the chalcone-derivative 8 possessing the Notch inhibitory activity at concentrations between 5–10 µm. It has been demonstrated that this inhibitor suppressed cell growth of several human T‑ALL cell lines, including DND41, KOPTK1 and TALL-1. This suppression is connected with the activation of apoptosis and increased expression of p27. Moreover, G1 cell cycle arrest has also been detected. Importantly, compound 8 inhibited Notch signaling without interfering with S2 and S3 proteolytic cleavages, depending on ADAM and GS, respectively. Nevertheless, this requires further investigation and development [33].

Extensive research over the past half a century indicates that Notch also plays a very significant role in the pathogenesis of B‑cell malignancies. Alterations in the Notch pathway have been observed in Hodgkin’s lymphoma, Burkitt’s lymphoma, B‑cell chronic lymphatic leukemia, diffuse large B‑cell lymphoma, primary effusion lymphomas related to Kaposi’s sarcoma herpes virus infection and multiple myeloma (MM) [34].

The possibility that Notch could play a significant role in human breast cancer development comes from studies on mouse mammary tumor virus-induced cancer. This study demonstrated that Notch4 showed an ability to transform mammary epithelium in vitro and in vivo. Moreover, an activated form of this receptor slowed ductal growth and perturbed lobular outgrowth prior to tumor formation [35].

The majority of breast cancers express estrogen, progesterone receptors (ER,PR), HER2 and/or ErbB-2 [36]. Zardawi and colleagues demonstrated that increased expression of Notch1 was an early event in both murine and human breast tumor development. Importantly, there was a connection between the high level of Notch1 and the HER-2 molecular subtype of breast cancer [37]. In this context, the study of Li et al., who found that Notch1 activity was not a characteristic feature of primary ductal breast carcinoma, appears to be very interesting. However, the high level of Notch1 has been significantly correlated with poorer differentiation of breast and prostate tumors [38]. Rizzo et al. reported that 80% of epithelial hyperplasias of usual type (HUTs), 67% of ductal carcinomas in situ (DCISs), 89% of invasive ductal carcinomas (IDCs) and 57% of invasive lobular carcinomas (ILCs) revealed high expression of Notch1, while expression in the non-pathological samples has been characterized as low or negative [39]. Mittal et al. also revealed the high level of Notch expression in IDC in comparison to normal tissues [40]. Bolos et al. demonstrated that ectopic expression of the NICD1 in the MCF-7 breast adenocarcinoma cell line has been associated with a reduction in the E‑cadherin level. Increased migratory and invasive abilities have also been detected in MDA-MB-231 cells. Notch1 activation in the mouse mammary gland was responsible for the formation of papillary tumors characterized by enhanced expression of Hes1 and Hey1, and delocalized E‑cadherin expression. Interestingly, the growth of subcutaneous xenografts generated with MCF-7 cells was boosted after NICD1 activation in a cell-autonomous manner. This may indicate that Notch1 is a key player in epithelial-to-mesenchymal transition (EMT) in breast cancer [41]. Shao et al. obtained similar results. These researches revealed that activation of JAG1 was also connected with EMT induction. Probably, this event was also related to upregulation of NICD1 [42]. It is worth noting that Notch1 played a vital role in regulation of EMT in a Slug-dependent manner. The luciferase reporter assay further demonstrated that Notch1 positively regulated Slug expression by enhancing Slug promoter activity. Notch1 might downregulate Slug to maintain the epithelial phenotype and inhibit the migration and invasion of breast cancer cells [42]. Knockdown of Notch1 significantly inhibited cell proliferation, migration and colony formation. In addition, downregulation of Notch1 caused cell cycle arrest in the G0/G1 phase, but promoted apoptosis under in vitro conditions. To further verify this mechanism, it was found that activation of Notch1 could upregulate the expression of cyclin D1, survivin and Bcl-xL. Therefore, knockdown of Notch1 arrested the cell cycle at G0/G1. This molecular event is probably cyclin D1-dependent and the inhibition of apoptotic cell death might be related to enhanced expression of survivin and Bcl-xL. Li et al. suggested that Notch-induced cell invasive growth might be associated with upregulation of MMP-9, MMP-2 and VEGF. Notch1 signaling might promote breast cancer cell invasion due to the activation of NF-κB and its downstream target genes including MMP-2, MMP-9 and VEGF [43].

The presence of JAG1 in primary lesions indicated a higher risk of metastatic relapse. JAG1 expression correlated with vascular invasion and lymph node involvement [44]. Dickson et al. also demonstrated that patients with high levels of JAG1 had a worse outcome in comparison to those with low levels of expression. This elevated expression of Notch ligands has been associated with the metastatic ability of breast cancer cells [45]. Moreover, it has been demonstrated that high expression of JAG1 shows a correlation with breast cancer metastasis [46, 47]. The analysis of the gene expression showed JAG1 to be one of 17 cytokine/ligand genes overexpressed in bone metastasis in comparison to liver, brain and lung metastasis, implying a potential organ-specific function for tumor-derived JAG1 in metastatic colonization of the bone [48]. Knockdown of JAG1 in breast cancer cells suppressed bone metastatic ability without impacting on the growth of cultured cells and primary mammary tumors. It was thought that this pro-metastatic function of JAG1 was mediated by two mechanisms: Firstly, by inducing osteoblasts to release IL-6, thereby affecting the growth of cancer cells; and secondly, by stimulating severe osteolysis, which is associated with space formation for metastasis [49].

Aberrant activation of the Notch signaling pathway has been associated with high grade ovarian carcinoma and carcinogenesis. Moreover, it plays a crucial role in chemoresistance in ovarian cancer patients [50]. The high level of Notch3 accompanied by a positive correlation with the expression of JAG1 and JAG2 has been detected in serous ovarian carcinomas. In addition, overexpression of Notch3 has been significantly correlated with advanced stage, lymph node metastasis, chemoresistance and poor overall survival [51]. In recurrent ovarian carcinoma enhanced expression of nuclear Notch3 also showed a link ro poor clinical outcome [52, 53]. The connection between the expression of Notch3 and increased resistance to chemotherapy, which remains a major obstacle to successful treatment, should also be mentioned here. Ectopic expression of Notch3 following transduction with NICD3 in ovarian surface epithelium and low-grade serous ovarian cancer cells led to increased resistance to carboplatin in vitro. In contrast, shRNA-mediated knockdown of Notch3 in OVCAR3 cells resulted in higher sensitivity to carboplatin compared with OVCAR3 cells transduced with a non-specific control shRNA [52]. Gupta et al. demonstrated that activation of Notch3 in OVCA429 cells was responsible for a fibroblast-like morphology and induction of the smooth muscle α‑actin, Slug and Snail. Interestingly, decreased expression of E‑cadherin has also been observed. This may suggest that Notch3 activation generates EMT in OVCA429 cells. Moreover, Notch3 activation rendered OVCA429 cells more resistant to carboplatin-induced cytotoxicity and impaired carboplatin-induced apoptosis [54]. Interesting results have been obtained by Kang et al., who demonstrated that Notch alteration impaired migration and angiogenesis and enhanced apoptotic cell death in paclitaxel-resistant cancer cells. The treatment of cells with Notch3 siRNA and gamma-secretase inhibitors (GSIs) was associated with arresting cells in the G2/M. At the same time, enhanced expression of proapoptotic proteins has been revealed [55].

The role of Notch1 in ovarian cancer was first described by Hopfer et al., who evaluated Notch mRNA expression in ovarian adenocarcinoma, borderline tumors and adenomas. They revealed more abundant expression of JAG2 and DLL1 in adenocarcinomas compared with adenomas. Interestingly, the stable transfection of A2780 cells with the NICD1 enhanced cell proliferation and increased colony-formation ability [56]. The high level of Notch1 has also been revealed in ovarian cancer cell lines including OVCAR3, SKOV3 and CaOV3. siRNA downregulation of NICD1 in those cells resulted in inhibition of cell proliferation. Notch1 also seems to be a very interesting factor in the context of chemoresistance [57‐60]. Dysregulation of miRNA alters a network of functional targets and signaling pathways resulting in acquired chemoresistance in human cancers. Liu and coworkers demonstrated that increased expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity. Conversely, re-expression of miR 199b-5p and siRNA-mediated JAG1 knockdown or treatment with GSI attenuated enhanced cisplatin-mediated cell cytotoxicity. It seems, therefore, that the epigenetic silencing of miR-199b-5p is significantly associated with acquired chemoresistance in ovarian cancer cells through the activation of JAG1-Notch1 [61]. The study by Zhou et al. provides evidence that downregulation of miR-449a could enhance ovarian cancer cell proliferation and induce cisplatin resistance. Importantly, inhibition of Notch1 signaling by overexpression of miR-449a could sensitize chemoresistant ovarian cancer cells to cisplatin-induced cytotoxicity. It may suggest that the miR-449a/Notch1 axis plays a significant role in the development of ovarian cancer [62]. Liang et al. revealed the role of miR-433. They reported that its overexpression significantly inhibited the migration and invasion of cancer cells, but had no significant effect on cell proliferation. Notch1 was a direct target of miR-433 and its downregulation inhibited the invasion of cancer cells [63].

Results of Alniaimi et al. demonstrated that Notch2 seems to be a tumour suppressor in ovarian carcinogenesis. The high expression of Notch2 mRNA has been significantly associated with poor PFS for all ovarian cancer patients, especially in grade II ovarian cancer. Interestingly, in serous and endometrioid cancer patients high expression of this mRNA was not correlated to poor PFS and OS [64].