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and carries a worse prognosis. Overexpression of Sox-9 leads HCC. Moreover, given the pan-inhibition nature of GSIs, the
to a transition to HPC-type activity and consequently less toxicity profiles are relatively disconcerting given the off
differentiated cell types. [73] target effects, especially intestinal adverse effects specifically
through down regulation of Notch-1 and -2 isoforms. [29]
As we advance our understanding of the Notch signaling
pathway, initial studies including several early phase I clinical As a result of the nonspecific inhibition of GSIs, alternative
trials are underway in various stepwise components within strategies should be considered. One particular area of
the pathway. Given the intricacy of the canonical Notch increasing interest is the use of monoclonal antibodies
pathway, it comes as no surprise that there are multiple and decoys at both the ligand and receptor sites. Antibody
avenues to target Notch signaling [Figure 1]. Targeting either and decoy (competitive antagonist) application has a more
the ligands and/or the receptors, inhibiting cleavage of the specific efficacy, thus limiting the dose-escalated toxicities
active NICD, and preventing transcription of downstream and potentially providing a concentrated result. There are
targets are the major targeted aims in Notch mediation. multiple monoclonal antibodies currently being tested in
In this review, we will briefly discuss the most common preclinical studies. Notch-1 receptor antibodies have shown
techniques aimed at inhibiting Notch signaling. promising results. [78,79] Both Notch-2 and Notch-3 antibodies
have transitioned to phase I clinical trials. [79,80] In addition
The most studied area is inhibition of GSI and the subsequent to Notch receptor blockade, antibodies against Notch
release of NICD. In fact, GSI examination is not inclusive to signaling ligands have been investigated. Delta-like ligand
HCC. Rather, the extensive research of Notch signaling in four antibodies have shown interesting results from multiple
neural development has led to GSI application for Alzheimer’s avenues. [58,81,82] Similarly, decoys provide excellent Notch
disease. There has been countless preclinical and phase inhibition and act as a competitive antagonist either at the
[74]
I clinical trials examining the efficacy and effectiveness of Notch receptor or the ligand binding sites. Notch-1 decoys
GSI mediation in many cancer types. [75-77] Unfortunately, have been studied as well as Jagged-1 ligand decoys. [83,84]
to date, there is no phase I evidence of the role of GSIs in Finally, prevention of NICD-mediated transcription is a novel
process to modulate carcinogenesis. Peptides that block
the transcription of NICD provide interesting applications
to Notch signal inhibition. In addition to this review
[75]
of potential Notch mediation through the alteration of
multiple events, Espinoza and Miele recently compiled
[75]
a comprehensive table and analysis including a majority
of current preclinical and clinical studies using a myriad of
Notch inhibitors that further details the current effectiveness
of Notch alteration.

Traditional mechanisms of Notch inhibition have and will
continue to be thoroughly investigated; however, there is
growing interest in targeted gene inhibition, more specifically
in the context of Notch signaling. Historically, AGS and
the mechanism of hepatogenesis were further delineated
through Notch-2 gene manipulation; therefore, an approach
to targeting specific genes within the Notch pathway may
Figure 1: Canonical Notch signaling pathway and potential sites of inhibition. provide additional support in lieu of traditional Notch
The Notch pathway is primed through cell-to-cell interaction distinguishing it inhibitors. There are several approaches to gene silencing,
from other regulatory pathways. Following ligand secretion from a transmitting two of which are frequently used and include using a
cell, the ligand binds to one of the four Notch receptors on the receiver cell.
Ligand-receptor binding facilitates the cleavage of the intracellular component small hairpin RNA or short hairpin RNA (shRNA) or siRNA.
of the transmembrane receptor via -secretase. Successful cleavage activates Mao et al. reported that the shRNA mediated knock-down
[85]
the Notch intracellular component domain (NICD) which translocates to the of Notch-1 inhibited the breast cancer cell line MCF-7’s
nucleus where is regulated a host of transcription factors. Given the intricacy of
the pathway, there are multiple key regulatory steps poised for targeted therapy. proliferation and induced cell apoptosis through multiple
First, monoclonal antibody and decoy administration at both the Notch ligand and mechanistic actions. One in particular, the down regulation
receptor is in preliminary, preclinical investigation. Second, -secretase inhibitors of the anti-apoptotic protein nuclear factor-kappa B, proved
are the most studied target within the pathway; however, there is limited data
within hepatocellular carcinoma. Finally, inactivating transcriptional peptides are effective and enhanced the anti-tumorigenic effect when
a novel trend focused on inhibiting the canonical transcription mediated by NICD combined with traditional chemotherapeutic agents such

14 Hepatoma Research | Volume 1 | Issue 1 | April 15, 2015