The importance of NOTCH signaling in cell growth, differentiation and survival is well established (Artavanis-Tsakonas et al., 1999
). However, NOTCH signaling is highly cell-context dependent, with the potential to activate or inhibit a range of different downstream pathways (Radtke and Raj, 2003
). It is likely that the effects of NOTCH signaling are specifically determined by which target genes, such as HES1-7, HERP1-3 and DTX1-4, are activated in a specific cell context. The regulation among these direct target genes and NOTCH receptors is critical for maintaining proper NOTCH signaling in specific cell environment. However, the mechanisms that regulate NOTCH target gene activation and NOTCH receptor stability are not fully understood. The present study reveals a molecular mechanism that regulates NOTCH signaling through reciprocal regulation of the NOTCH target genes HES1 and DTX1 in human cells. More importantly, our analysis has clinical implications for the use of NOTCH inhibitors for osteosarcoma and other cancers.
Our present studies suggest a novel mechanism of reciprocal regulation between the NOTCH target genes HES1 and DTX1: 1) DTX1 negatively regulates NOTCH/HES1 signaling, and 2) HES1 inhibits DTX1 mRNA expression in osteosarcoma cells. Based on our analysis, we propose a model for the regulation of NOTCH signaling by HES1 and DTX1 (). In our model, DTX1, a CSL-dependent NOTCH target gene that also has NOTCH-independent mechanisms of expression, provides negative feedback for NOTCH signaling by targeting ICN1 for ubiquitination and proteasome-mediated degradation. Since our system used a genetically-defined ICN fragment, we know that this mechanism can act on the GSI-cleaved fragment of Notch1. It is possible that DTX1 might also be able to act on full-length Notch1 as well. HES1 regulates this negative feedback through direct transcriptional repression of the DTX1 promoter. Consequently, the impact of NOTCH signaling is affected by a balance of HES1 and DTX1. In osteosarcoma cells, we previously showed that NOTCH signaling, and specifically HES1, promotes invasiveness and metastasis in vivo
(Zhang et al., 2008
). Inhibition of NOTCH signaling by γ–secretase inhibitor (GSI) blocked osteosarcoma cell invasion in OS187 cells. However, another osteosarcoma cell line, COL, is resistant to GSI treatment (Zhang et al., 2008
). In this study, we suggest that invasion may be regulated by the reciprocal inhibition of HES1 and DTX1. If an osteosarcoma cell has high HES1 and low DTX1, such as OS187 cells (Zhang et al., 2008
), GSI or other NOTCH inhibitors may repress HES1 expression, and tumor cell invasion. However, if an osteosarcoma has high DTX1 and lower HES1, such as COL, GSI may not suppress tumor cell invasion.
If the regulation of Notch signaling in humans is similar to drosophila, then the model may be even more complex. Matsuno et al. have shown that dominant negative Deltex can function upstream of ICN but downstream of full-length Notch, through binding of the proline-rich domain with the ankyrin repeats of Notch (Matsuno et al., 2002
). Their further studies demonstrated that Deltex could mediate ligand-independent activation of Notch (Hori et al., 2004
), and that the level of Deltex activity might dictate whether Deltex mediates cleavage and activation of Notch or ubiquitin-mediated lysosomal degradation of Notch and/or ICN (Wilkin et al., 2008
). Our data in humans would provide direct mechanistic support for such a model in osteosarcoma.
In our study, the C-terminal region of HES1 was necessary for transcriptional repression of DTX1, but the WRPW motif was dispensible. Previous research has shown that WRPW domain is required for HES1 recruiting the corepressor TLE (Groucho in Drosophila
) to the target gene promoter (Cinnamon and Paroush, 2008
; Struhl and Adachi, 1998
). Our data suggests that the Groucho/TLE binding to the WRPW motif is not essential for HES1 mediated repression of DTX1 in osteosarcoma. Further studies are needed to determine if other corepressors are also involved in the inhibition of DTX1 promoter, e.g. CtBP.
HES1 functions as transcription repressor by forming homodimers or heterodimers with HERP (Iso et al., 2003
). dnHES1 may also form heterodimers with HERP protein and regulate HERP function. We found that dnHES1 can upregulate DTX1 gene expression even in COL cells which have low endogeous HES1. In this case, dnHES1 might regulate DTX1 by affecting other interacting partners for HES1 such as HERP2
, which is present in COL (Zhang et al., 2008
DTX1, while clearly a NOTCH target gene, has also been shown to have NOTCH regulatory functions. In Drosophila
, the non-visual β–arrestin (Kurtz) is required for mediating the interaction of Dx (Drosophila
homolog of DTX1) and NOTCH (Mukherjee et al., 2005
). However, DTX1 prevented ICN1 from recruiting coactivators, limiting ICN1 CSL activation in mammalian cells (Jackson et al., 2000
). Protein structure studies illustrate that the two WWE repeats in N-terminal domain of Dx form heterodimers with the ankyrin domain of NOTCH (Zweifel et al., 2005
). Here we show that DTX1 physically binds to ICN1, inducing ubiquitination of ICN1. Similar to the Dx effects on Notch signaling (Mukherjee et al., 2005
), human DTX1 inhibition on ICN1 is not very strong. However, we have not explored the role of human β–arrestin in the DTX1 and ICN1 regulation in human cells. Further studies are needed to examine whether β–arrestin will enhance the DTX1 inhibitory effects on ICN1 in human cells.
NOTCH function in cancers is complex, since NOTCH signaling can be oncogenic or act as a tumor suppressor in different cancer types. For those cancers in which NOTCH functions as an oncogene, NOTCH inhibitors may provide therapeutic benefit. Based on our results, we suggest that in these malignancies the potential benefit of a NOTCH inhibitor may depend on the relative expression of both HES1 and DTX1. GSI might only benefit osteosarcoma patients with tumors expressing high HES1 and low DTX1, but not those with high DTX1. Low DTX1 expression may be useful as a marker to select osteosarcoma patients who could benefit from NOTCH inhibitor treatment. This regulatory mechanism may exist in other types of cancer and should be evaluated, since NOTCH inhibitors such as GSI are being evaluated in clinical trials.
In summary, our study identifies a novel regulatory mechanism of NOTCH signaling by reciprocal inhibition of its own target genes, HES1 and DTX1 in human cells. Our data suggest that DTX1 also plays a role in regulation of osteosarcoma invasiveness, likely through NOTCH/HES1. We believe that this regulation is important not only for our understanding of NOTCH signaling itself but also for its implication on the selection of patients for clinical trials related to NOTCH for cancer.