Multiple functions of Notch signaling in self-renewing organs and cancer

In recent years a substantial body of evidence has accumulated to support the notion that signaling pathways known to be important during embryonic development play important roles in regulating self-renewing tissues. Moreover, the same pathways are often deregulated during tumorigenesis due to mutations of key elements of these pathways. The Notch signaling cascade meets all of the above-mentioned criteria. We discuss here the pleiotropic roles of the Notch signaling pathway in three different self-renewing organs (intestine, hematopoietic system and skin) and how its deregulation is involved in tumorigenesis.     

Notch Pathway Is Activated by MAPK Signaling and Influences Papillary Thyroid Cancer Proliferation

Mutually exclusive genetic alterations in the RET, RAS, or BRAF genes, which result in constitutively active mitogen-activated protein kinase (MAPK) signaling, are present in about 70% of papillary thyroid carcinomas (PTCs). However, the effect of MAPK activation on other signaling pathways involved in oncogenic transformation, such as Notch, remains unclear. In this study, we tested the hypothesis that the MAPK pathway regulates Notch signaling and that Notch signaling plays a role in PTC cell proliferation. Conditional induction of MAPK signaling oncogenes RET/PTC3 or BRAF^T1799A in normal rat thyroid cell line mediated activation of Notch signaling, upregulating Notch1 receptor and Hes1, the downstream effector of Notch pathway. Conversely, pharmacological inhibition of MAPK reduced Notch signaling in PTC cell. Thyroid tumor samples from transgenic mice expressing BRAF^T1799A and primary human PTC samples showed high levels of Notch1 expression. Down-regulation of Notch signaling by γ-secretase inhibitor (GSI) or NOTCH1 RNA interference reduces PTC cell proliferation. Moreover, the combination of GSI with a MAPK inhibitor enhanced the growth suppression in PTC cells. This study revealed that RET/PTC and BRAF^T1799A activate Notch signaling and promote tumor growth in thyroid follicular cell. Taken together, these data suggest that Notch signaling may be explored as an adjuvant therapy for thyroid papillary cancer.     

Notch signaling in melanoma: interacting pathways and stromal influences that enhance Notch targeting

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling cascade. Notch was first described in the early 1900s when a mutant Drosophila showed notches on the wing margins. Studies of the role of Notch signaling have ever since flourished, and the pleiotropic nature of the Notch gene is now evident. Indeed, the Notch signaling pathway plays key roles in cell fate decisions, tissue patterning, and morphogenesis during development. However, deregulation of this pathway can contribute to cell transformation and tumorigenesis. Several reports have now highlighted the role of Notch signaling in a variety of malignancies where Notch can either be an oncogene or a tumor suppressor depending on the cell context. Here, we summarize the major components of Notch signaling with an aim to emphasize the contribution of deregulated Notch signaling in melanomagenesis.     

Elucidation of rutin’s role in inducing caspase-dependent apoptosis via HPV-E6 and E7 down-regulation in cervical cancer HeLa cells

Over the recent few years rutin has gained wider attention in exhibiting inhibitory potential against several oncotargets for inducing apoptotic and antiproliferative activity in several human cancer cells. Several deregulated signaling pathways are implicated in cancer pathogenesis. Therefore we have inclined our research towards exploring the anticancerous efficacy of a very potent phytocompound for modulating the incontinent expression of these two crucial E6 and E7 oncogenes. Further, inhibitory efficacy of rutin against human papillomavirus (HPV)-E6 and E7 oncoproteins in cervical cancer has not been elucidated yet. This research addresses the growth inhibitory efficacy of rutin against E6 and E7 oncoproteins in HeLa cells, which is known to inactivate several tumor suppressor proteins such as p53 and pRB. Rutin treatment exhibited reduced cell viability with increased cell accumulation in G₀/G₁ phase of cell cycle in HeLa cell lines. Additionally, rutin treatment has also led to down-regulation of E6 and E7 expression associated with an increased expression of p53 and pRB levels. This has further resulted in enhanced Bax expression and decreased Bcl-2 expression releasing cytochrome c into cytosol followed by caspase cascade activation with cleavage of caspase-3, caspase-8 and caspase-9. Further, in silico studies have also supported our in vitro findings by exhibiting significant binding energy against selected target oncoproteins. Therefore, our research findings might recommend rutin as one of the potent drug candidate in cervical cancer management via targeting two crucial oncoproteins associated with viral progression.     

ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumorigenesis

The ErbB-2 receptor is overexpressed in roughly 30% of human breast cancers. Moreover, approximately 50% of breast cancers are positive for high-risk human papillomaviruses (HPVs), specifically types 16 and 18. Recently, we reported that ErbB-2 cooperates with E6/E7 oncoproteins of HPV type 16 to induce neoplastic transformation of human normal oral epithelial cells. We also demonstrated that E6/E7 of HPV type 16 converts non-invasive breast cancer cells to an invasive form. In order to investigate the effect of ErbB-2/E6/E7 cooperation in breast carcinogenesis, we generated double transgenic mice carrying ErbB-2 and E6/E7 of HPV type 16 under mouse mammary tumor virus (MMTV) and human keratin 14 promoters, respectively. Within six months, these double transgenic mice developed large and extensive invasive breast cancer in comparison to ErbB-2 or E6/E7 singly transgenic mice. Histological analysis of ErbB-2/E6/E7 transgenic mice tumors showed the presence of invasive breast carcinomas. However, the breast tissues from ErbB-2 and E6/E7 transgenic mice showed only in-situ cancer and normal mammary phenotype, respectively. In parallel, we examined the cooperation effect of ErbB-2 and E6/E7 in the human breast cancer cell line, BT20; in comparison to ErbB-2 and E6/E7 alone as well as wild type cells, we found that ErbB 2/E6/E7 together stimulate colony formation and cell migration in the BT20 cell line. Furthermore, we found that β-catenin is constitutively phosphorylated by c-Src and consequently trans-located to the nucleus in ErbB-2/E6/E7-breast cancer cells. These findings provide evidence that the ErbB-2 receptor cooperates with high-risk HPVs in breast tumorigenesis via β-catenin activation.     

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.     

Notch signaling suppresses p38 MAPK activity via induction of MKP-1 in myogenesis

Cross-talks among intracellular signaling pathways are important for the regulation of cell fate decisions and cellular responses to extracellular signals. Both the Notch pathway and the MAPK pathways play important roles in many biological processes, and the Notch pathway has been shown to interact with the ERK-type MAPK pathway. However, its interaction with the other MAPK pathways is unknown. Here we show that Notch signaling activation in C2C12 cells suppresses the activity of p38 MAPK to inhibit myogenesis. Our results show that Notch specifically induces expression of MKP-1, a member of the dual-specificity MAPK phosphatase, which directly inactivates p38 to negatively regulate C2C12 myogenesis. The Notch-induced expression of MKP-1 is shown to depend on RBP-J. Moreover, inhibition of MKP-1 expression by short interfering RNA suppresses p38 inactivation and partially rescues the negative regulation of myogenesis. These results reveal a novel cross-talk between the Notch pathway and the p38 MAPK pathway that is mediated by Notch induction of MKP-1.     

Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway

Activated Notch1 (AcN1) alleles cooperate with oncogenes from DNA tumor viruses in transformation of epithelial cells. AcN1 signaling has pleiotropic effects, and suggested oncogenic roles include driving proliferation through cyclin D1 or the generation of resistance to apoptosis on matrix withdrawal through a phosphatidylinositol 3-kinase (PI3K)-PKB/Akt-dependent pathway. Here, we extend the antiapoptotic role for AcN1 by showing inhibition of p53-induced apoptosis and transactivation. Chemical inhibitors of the PI3K pathway block AcN1-induced inhibition of p53-dependent apoptosis and nuclear localization of Hdm2. We show that expression of wild-type p53 does not inhibit synergistic transformation by AcN1 and human papillomavirus E6 and E7 oncogenes. We suggest that activation of Notch signaling may serve as an additional mechanism to inhibit wild-type p53 function in papillomavirus-associated neoplasia.     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

Role of nodal signaling and the microenvironment underlying melanoma plasticity

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.