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Page 8 of 18 Lee et al. J Cancer Metastasis Treat 2021;7:27 https://dx.doi.org/10.20517/2394-4722.2021.58

Despite JQ1’s effective inhibition of tumor growth via suppression of MYC transcription, it showed no
apparent inhibitory effects on tumor invasion and metastasis. This suggested that there were other
oncogenic events to drive invasion and metastasis for the ATC progression. The MAPK-MEK signaling
pathway is frequently upregulated in human ATC and is related to the ATC progression [126-128] . An MEK
inhibitor, trametinib, was therefore used as a combined treatment with JQ1 to test this hypothesis in two
[129]
human ATC cells, THJ-11T and -16T . Remarkably, although either JQ1 alone or trametinib alone
showed only partial effects, the combined treatment totally blocked proliferation of the ATC cells.
Combined treatment downregulated MYC expression much more than each single treatment did, leading to
suppression of pro-survival regulators and induction of pro-apoptotic regulators to cooperatively induce
apoptosis. In xenograft studies, while each single treatment only partially inhibited growth of either THJ-
11T or -16T-induced tumors, the combined treatment near completely (> 90%) blocked the tumor growth.
This dramatic inhibition of tumor growth by the combined treatment occurred through synergistic
suppression of MYC, which induced apoptotic regulators thereby markedly promoting tumor apoptosis.
The underlying mechanism that the combined treatment synergistically suppressed MYC expression was
further studied. Chromatin immunoprecipitation (ChIP) assay was used to probe the effects of JQ1 and
trametinib on the binding of BRD4 to the MYC promoter in THJ-11T and -16T cells. As shown in Figure 2,
JQ1, trametinib, and the combined treatment inhibited the BRD4 binding to the MYC promoter by 55%,
42%, and 69%, respectively, compared to the control (vehicle-treated cells), in THJ-11T cells [Figure 2C-i].
Similar efficacy profiles of the three treatments in the BRD4 binding suppression were observed in THJ-16T
cells (46%, 36%, and 64% by JQ1, trametinib, and the combined treatment, respectively, Figure 2C-ii). These
data indicated that JQ1 and trametinib functioned, at least in part, to inhibit the BRD4 binding to histone
acetyl-lysine sites across the chromatin. Further, these two inhibitors could synergistically suppress MYC
transcription via cooperative actions on chromatic modifications [Figure 2A and B].

The efficacy of combined treatment with BET and MEK inhibitors was further demonstrated by using the
second generation of a BET inhibitor. A new BET inhibitor PLX51107 (PLX) has demonstrated more
[130]
favorable pharmacokinetic profiles than JQ1 and other BET inhibitors . It has been under clinical trials
for various solid tumors and hematological malignancies. PD0325901 (PD) is a MEK inhibitor that has also
[131]
been evaluated in clinical trials for several cancers . PLX and PD individually could suppress proliferation
of both THJ-11T and -16T cells, but together exhibited synergistic inhibition. In mouse xenografts derived
from the ATC cells, the combined treatment nearly completely blocked in vivo tumor growth. PD effectively
reduced MEK-ERK signaling, and this inhibition was further augmented by the combined treatment with
PLX in the ATC cells and tumors. Notably, PLX and PD synergistically attenuate MYC transcription to
induce p27 for the tumor suppression. They also cooperated to activate pro-apoptotic regulators to induce
apoptosis. These data indicated cooperation of PLX and PD that block BRD4 binding to histone acetyl-
lysine sites on the promoter of the MYC gene. These collaborative actions could converge to induce
epigenetic modifications to suppress MYC transcription. The efficacy of combined treatment was clearly
demonstrated by using two different sets of BET and MEK inhibitors. These findings clearly demonstrated
that epigenetic modifications on chromatin is a viable and effective approach for the treatment of ATC.

Steroid hormone nuclear receptor coactivators
The steroid hormone nuclear receptor coactivators (SRCs: SRC-1, SRC-2, and SRC-3) are important
transcriptional coactivators discovered initially for the regulation of the transcriptional activity of the
nuclear receptor superfamily. Subsequently, other transcription factors - including STATs, P53, RB, E2F1,
hypoxia inducible factor-1 (HIF-1), Smads, and nuclear factor-κB (NF-κB) - were also found to be
[132]
modulated by SRCs . Upon ligand (hormone) binding, the ligand-bound nuclear receptors (NRs) open
their coactivator-binding motifs in their ligand-binding domains and recruit SRCs to the enhancer sites of
NR-target genes. SRCs further recruit other common transcriptional coactivators such as CBP/p300 and