The Notch signaling pathway in esophageal adenocarcinoma.

The Notch signaling pathway plays a critical role in embryonic development, self-renewal of stem cells, and carcinogenesis. Aberrant Notch signaling has been linked to a wide variety of cancers, and can either suppress or promote tumors depending on the cell type and the context. Increasingly it is being realized that Notch signaling not only involves in the pathogenesis and development of esophageal adenocarcinoma (EAC), it also promotes the growth of EAC cells and also involved in the maintenance of EAC cancer stem cells. The efficacy of gamma-secretase inhibitor (GSI) in EAC treatment could have a major impact on easing the burden of this devastating disease. Therefore, it appears that inhibition of Notch sensitizes EAC cells to chemotherapeutic agents, which should lead to a better and more durable response to neoadjuvant chemotherapy (NAC). In this review, we bring to highlight how Notch plays a role in the development, tumorigenicity, and stemness of EAC cells, and how Notch signaling pathway could be a promising therapeutic target for the treatment of human EAC.

Induction of cyclin D1 transcription and CDK2 activity by Notch(ic): implication for cell cycle disruption in transformation by Notch(ic).

Notch genes encode a family of transmembrane proteins that are involved in many cellular processes such as differentiation, proliferation, and apoptosis. Although it is well established that all four Notch genes can act as oncogenes, the mechanism by which Notch proteins transform cells remains unknown. Previously, we have shown that transformation of RKE cells can be conditionally induced by hormone activation of Notch(ic)-estrogen receptor (ER) chimeras. Using this inducible system, we show that Notch(ic) activates transcription of the cyclin D1 gene with rapid kinetics. Transcriptional activation of cyclin D1 is independent from serum-derived growth factors and de novo synthesis of secondary transcriptional activators. Moreover, hormone activation of Notch(ic)-ER proteins induces CDK2 activity in the absence of serum. Upregulation of cyclin D1 and activation of CDK2 by Notch(ic) result in the promotion of S-phase entry. These data demonstrate the first evidence that Notch(ic) proteins can directly regulate factors involved in cell cycle control and affect cellular proliferation. Furthermore, nontransforming Notch(ic) proteins do not induce cyclin D1 expression, indicating that the mechanism of transformation involves cell cycle deregulation through constitutive expression of cyclin D1. Finally, we have identified a CSL [stands for CBF1, Su(H), and Lag-1] binding site within the human and rat cyclin D1 promoters, suggesting that Notch(ic) proteins activate cyclin D1 transcription through a CSL-dependent pathway.

Neoplastic transformation by Notch requires nuclear localization.

Notch proteins are plasma membrane-spanning receptors that mediate important cell fate decisions such as differentiation, proliferation, and apoptosis. The mechanism of Notch signaling remains poorly understood. However, it is clear that the Notch signaling pathway mediates its effects through intercellular contact between neighboring cells. The prevailing model for Notch signaling suggests that ligand, presented on a neighboring cell, triggers proteolytic processing of Notch. Following proteolysis, it is thought that the intracellular portion of Notch (N(ic)) translocates to the nucleus, where it is involved in regulating gene expression. There is considerable debate concerning where in the cell Notch functions and what proteins serve as effectors of the Notch signal. Several Notch genes have clearly been shown to be proto-oncogenes in mammalian cells. Activation of Notch proto-oncogenes has been associated with tumorigenesis in several human and other mammalian cancers. Transforming alleles of Notch direct the expression of truncated proteins that primarily consist of N(ic) and are not tethered to the plasma membrane. However, the mechanism by which Notch oncoproteins (generically termed here as N(ic)) induce neoplastic transformation is not known. Previously we demonstrated that N1(ic) and N2(ic) could transform E1A immortalized baby rat kidney cells (RKE) in vitro. We now report direct evidence that N1(ic) must accumulate in the nucleus to induce transformation of RKE cells. In addition, we define the minimal domain of N1(ic) required to induce transformation and present evidence that transformation of RKE cells by N1(ic) is likely to be through a CBF1-independent pathway.

A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel.

The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are related through a highly conserved domain of approximately 300 amino acids, called the Rel homology domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel homology domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.

Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2.

The Notch genes of Drosophila melanogaster and vertebrates encode transmembrane receptors that help determine cell fate during development. Although ligands for Notch proteins have been identified, the signaling cascade downstream of the receptors remains poorly understood. In human acute lymphoblastic T-cell leukemia, a chromosomal translocation damages the NOTCH1 gene. The damage apparently gives rise to a constitutively activated version of NOTCH protein. Here we show that a truncated version of NOTCH1 protein resembling that found in the leukemic cells can transform rat kidney cells in vitro. The transformation required cooperation with the E1A oncogene of adenovirus. The transforming version of NOTCH protein was located in the nucleus. In contrast, neither wild-type NOTCH protein nor a form of the truncated protein permanently anchored to the plasma membrane produced transformation in vitro. We conclude that constitutive activation of NOTCH similar to that found in human leukemia can contribute to neoplastic transformation. Transformation may require that the NOTCH protein be translocated to the nucleus. These results sustain a current view of how Notch transduces a signal from the surface of the cell to the nucleus.

Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma.

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Repression of the chicken c-rel promoter by vRel in chicken embryo fibroblasts is not mediated through a consensus NF-kappa B binding site.

To understand the regulation of expression of the chicken c-rel gene, we cloned genomic sequences upstream of the start site of transcription of c-rel. Sequence analysis shows that the c-rel promoter is a GC-rich promoter that lacks a TATA box. In addition, there are putative binding sites for several transcription factors, including an NF-kappa B consensus binding site. Primer extension showed that there is one major start site (site 1) for transcription in chicken embryo fibroblasts and two major start sites in a v-rel-transformed chicken spleen cell line. In transient assays using c-rel promoter sequences and the CAT reporter gene, we found that vRel repressed expression from the c-rel promoter. Other viral oncoproteins and a non-transforming v-rel deletion mutant did not repress the c-rel promoter. Repression occurred through sequences located within 125 bp of the start of transcription. However, mutation of the consensus NF-kappa B binding site did not affect the level of transcription from the c-rel promoter, nor did it interfere with repression by vRel, even though vRel could bind to the wild-type, but not the mutant, version of this sequence in vitro. These results suggest that the vRel protein can repress transcription through an indirect mechanism.

Notch(ic)-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation.

Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in many distinct cellular processes such as differentiation, proliferation and apoptosis. Notch function has been shown to be required both during development and in adult life. Moreover, several studies on spontaneous human tumors and in experimental models demonstrate that three of the four mammalian Notch genes can act as oncogenes. The mechanism by which Notch proteins induce neoplastic transformation is not known. In order to determine the early signaling events mediated by Notch during cellular transformation we constructed several inducible alleles of Notch(ic) by fusing portions of Nic to the hormone-binding domain of the estrogen receptor. Here we show that Notch(ic)-ER chimeras are conditionally activated by 4-Hydroxytamoxifen (OHT) in a dose-dependent manner. Clonal RKE cell lines expressing Notch(ic)-ER chimeras display hormone-dependent transformation in vitro. Transformation mediated by Notch(ic)-ER is reversible and chronic stimulation is necessary for the maintenance of the transformed phenotype. In response to hormone activation Notch(ic)-ER chimeras become hyperphosphorylated and accumulate in the nucleus of the cell; indicating that both phosphorylation and nuclear localization are required for Notch transforming activity.

The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts.

The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of a family of related transcription factors, which also includes the subunits of NF-kappa B and several other interacting cellular proteins. We show here that v-Rel specifically increased expression from a reporter plasmid containing multiple Sp1 binding sites approximately sixfold in chicken embryo fibroblasts (CEFs), even though v-Rel did not bind directly to these sites. v-Rel also increased expression from a reporter plasmid containing a human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in which the kappa B binding sites were mutated but which still contained intact Sp1 binding sites. The increase in Sp1-site transactivation does not precisely correlate with transformation by v-Rel since one non-transforming v-Rel mutant still induced expression from the Sp1 site-containing promoter. v-Rel appears to increase expression from Sp1 site-containing promoters by affecting the transactivation domain of Sp1, since v-Rel increased the activity of a Gal4-Sp1 fusion protein, which contains the Sp1 transactivation domain but lacks the Sp1 DNA-binding domain. As compared with v-Rel, c-Rel induced only a slight increase in expression from the reporter plasmid containing Sp1 sites. However, v-Ras and v-Src (but not v-Myb) induced increases in transcription from the reporter plasmid containing Sp1 sites to the same extent as v-Rel, but through pathways that appear to be independent from v-Rel. These results suggest that certain oncoproteins might increase transcription from many genes that contain Sp1 binding sites, and that this might be important for certain aspects of transformation by these proteins.

Cloning and expression of a chicken c-rel cDNA: unlike p59v-rel, p68c-rel is a cytoplasmic protein in chicken embryo fibroblasts.

We isolated and sequenced a 3727 bp clone of the c-rel proto-oncogene from a chicken embryo fibroblast (CEF) cDNA library. Sequence comparison to the retroviral oncogene v-rel showed conclusively that the v-rel protein is truncated at both the amino- and carboxy-termini as compared to the c-rel protein. In vitro transcription and translation of this clone yielded a 68,000 dalton polypeptide that co-migrated on SDS polyacrylamide gels with p68c-rel from avian spleen cells. We inserted this c-rel cDNA clone into an avian retroviral vector (pJD214c-rel), and over-expressed p68c-rel in CEF. Over-expression of p68c-rel did not induce morphological transformation of these cells. Unlike p59v-rel, which is a nuclear protein in CEF, indirect immunofluorescence showed that p68c-rel in JD214c-rel infected CEF is located exclusively in the cytoplasm of these cells, even though the sequence of p68c-rel showed that it contains a nuclear localizing sequence identical to the one previously identified in p59v-rel. Furthermore, the c-rel protein does contain a nuclear localizing sequence which can function in CEF since replacement of the v-rel nuclear localizing sequence with the homologous domain from c-rel resulted in a hybrid rel protein that was located in the nucleus in CEF. Mutant c-rel proteins, deleted of the carboxy-terminal sequences not present in p59v-rel, localized to the nucleus in CEF. Our results show that the carboxy-terminus of p68c-rel inhibits nuclear localization of the protein, and suggest that subcellular location may be a form of regulation of the activity of p68c-rel.

Characterization of a chicken cDNA encoding the retinoblastoma gene product.

We have isolated a chicken cDNA that encodes the retinoblastoma susceptibility gene product (RB). The predicted amino acid sequence of the chicken RB protein is highly similar to that of the mouse, human and Xenopus RB proteins in regions of known functions; however, chicken RB has distinct species-specific differences, including a shorter N-terminal region as compared to the mouse and human RB proteins. In vitro-translated chicken RB co-migrates on SDS-polyacrylamide gels with endogenous RB synthesized in transformed chicken spleen cells. Finally, chicken RB is located in the nucleus of chicken embryo fibroblasts when overexpressed from a retroviral vector.

The hedgehog processing pathway is required for NSCLC growth and survival.

Considerable interest has been generated from the results of recent clinical trials using smoothened (SMO) antagonists to inhibit the growth of hedgehog (HH) signaling-dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH-dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, skinny hedgehog (SKN) or dispatched-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently overexpressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand-dependent cancers.

It's T-ALL about Notch.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subset of ALL with poor clinical outcome compared to B-ALL. Therefore, to improve treatment, it is imperative to delineate the molecular blueprint of this disease. This review describes the central role that the Notch pathway plays in T-ALL development. We also discuss the interactions between Notch and the tumor suppressors Ikaros and p53. Loss of Ikaros, a direct repressor of Notch target genes, and suppression of p53-mediated apoptosis are essential for development of this neoplasm. In addition to the activating mutations of Notch previously described, this review will outline combinations of mutations in pathways that contribute to Notch signaling and appear to drive T-ALL development by 'mimicking' Notch effects on cell cycle and apoptosis.

Expression of JAGGED1 in T-lymphocytes results in thymic involution by inducing apoptosis of thymic stromal epithelial cells.

Proper development of the thymus and differentiation of T-lymphocytes requires cell-cell interactions between the developing T-lymphocytes and the thymic epithelia. The Delta/Serrate/Lag-2 (DSL)/Notch signal-transduction pathway is known to govern cell fate decisions required for proper development through direct cell-cell interactions. The functional consequences of specific DSL/Notch interactions during the development of a complex organ, such as the thymus, have not been thoroughly elucidated, however. In order to examine the role of DSL proteins during thymus development and T-lymphocyte differentiation, we targeted expression of JAGGED1 in T-lymphocyte progenitors via the control of the proximal lck promoter. Here, we report that expression of JAGGED1 in T cells causes premature involution of the thymus by directing thymic epithelial cells to undergo an apoptotic program. Adoptive transfer of JAGGED1 transgenic bone marrow into non-transgenic mice revealed that JAGGED1 expression on T cells does not alter T-cell differentiation, but is directly responsible for involution of the thymus. We propose that the phenotype of the lck-JAGGED1 transgenic mice is a direct result of specific DSL/Notch interactions and improper cell-to-cell signaling.

A conditional mutant of vRel containing sequences from the human estrogen receptor.

The mechanism by which the v-rel oncogene of the avian Rev-T retrovirus transforms chicken spleen cells is not known. We have created v-rel mutants that show conditional properties by fusing sequences encoding the ligand-binding domain of the human estrogen receptor (ER) in-frame at the 3' end of the v-rel oncogene. Two vRel-ER fusion proteins showed estrogen-dependent subcellular localization in chicken embryo fibroblasts (CEF): vRel-ER proteins were located in the cytoplasm of CEF in the absence of estrogen and were located in the nucleus of CEF in the presence of estrogen. Wild-type vRel was located in the nucleus of CEF in the presence or absence of estrogen. Mobility shift assays using extracts from infected CEF showed that the ability of vRel-ER to bind DNA was also dependent on estrogen. However, the ability of vRel-ER to repress transcription from kappa B site-containing promoters was not dependent on estrogen. Finally, we were able to isolate a vRel-ER-transformed avian spleen cell line whose growth is dependent on estrogen; this indicates that a vRel function is needed for both the initiation and the maintenance of the transformed state. The vRel-Er protein may be useful for determining genes controlled by vRel.

p105, the NF-kappa B p50 precursor protein, is one of the cellular proteins complexed with the v-Rel oncoprotein in transformed chicken spleen cells.

Active NF-kappa B-like transcription complexes are multimers consisting of one or two members of a family of proteins related to the c-Rel proto-oncoprotein. We have isolated a chicken cDNA encoding p105, the precursor protein for the p50 subunit of NF-kappa B. Sequence analysis shows that chicken p105 is approximately 70% identical to the mouse and human p105 proteins, containing the Rel homology domain in its N-terminal 370 amino acids and several ankyrinlike repeats in the C-terminal portion of the protein. The Rel homology domain is particularly highly conserved between chicken and mammalian p50, and an in vitro-synthesized, truncated chicken p105 protein, containing sequences that correspond to the predicted p50 protein, bound to a consensus kappa B site in an electrophoretic mobility shift assay. In v-Rel-transformed chicken spleen cells, v-Rel is found in high-molecular-weight complexes which include cellular proteins of approximately 124 kDa (p124) and 115 kDa (p115). Here we report that in vitro-produced p105 comigrates with p124 from v-Rel-transformed spleen cells and that p105 and p124 appear to be identical by partial proteolytic mapping with V8 protease. Furthermore, both p105 and p50 can complex directly with v-Rel and chicken c-Rel in vitro. However, in vitro association with p105 by v-Rel does not necessarily correlate with transformation, since one nontransforming v-Rel mutant can associate with p105 in vitro.

Suppression of erythroid but not megakaryocytic differentiation of human K562 erythroleukemic cells by notch-1.

The Notch signal transduction pathway is a highly conserved regulatory system that controls multiple developmental processes. We have established an erythroleukemia cell model to study how Notch regulates cell fate and erythroleukemic cell differentiation. K562 and HEL cells expressed the Notch-1 receptor and the Notch ligand Jagged-1. The stable expression of the constitutively active intracellular domain of Notch-1 (NIC-1) in K562 cells inhibited erythroid without affecting megakaryocytic maturation. Expression of antisense Notch-1 induced spontaneous erythroid maturation. Suppression of erythroid maturation by NIC-1 did not result from down-regulation of GATA-1 and TAL-1, transcription factors necessary for erythroid differentiation. Microarray gene expression analysis identified genes activated during erythroid maturation, and NIC-1 disrupted the maturation-dependent changes in the expression of these genes. These results show that NIC-1 alters the pattern of gene expression in K562 cells leading to a block in erythroid maturation and therefore suggest that Notch signaling may control the developmental potential of normal and malignant erythroid progenitor cells.