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Page 10 of 18 Lee et al. J Cancer Metastasis Treat 2021;7:27 https://dx.doi.org/10.20517/2394-4722.2021.58

Although transcriptional coactivators are difficult to target because of their large size and disordered
[144]
structures , a new generation SRC-3 inhibitor-2 (SI-2) was developed through cellular function-based
high-throughput screening. SI-2 selectively targeted breast cancer cells through inhibition of SRC-3
[145]
transcriptional activities . On the basis of known complex transcriptional oncogenic changes observed in
ATC [22,23,146] and the critical role of SRC-3 for transcriptional regulation, the expression of SRC-3 was
examined during human thyroid cancer progression from normal, through DTC (FTC and PTC), to
ATC . Comparison of the SRC-3 protein abundance among human normal thyroid tissue [Figure 3A-I-a],
[147]
FTC [Figure 3A-I-b], papillary thyroid cancer (PTC) [Figure 3A-I-c], and ATC [Figure 3A-I-d-f] shows that
SRC-3 is clearly higher in ATCs than in normal thyroid tissues, FTC, and PTC [Figure 3A-II]. Quantitative
analysis shows that 54.6% of ATC cells were positive for SRC-3 vs. only 18.6% of PTC cells, 13.9% of FTC
cells, and 18.3% of normal thyroid cells [Figure 3A-III]. Of note, further investigation for co-expression of
SRC-3 and Ki-67 (a proliferation marker), clearly demonstrated a strong positive correlation (r = 0.8447, P <
0.0001) between SRC-3 and Ki-67 expression in human ATC tissues, suggesting that hyperactive
transcriptional responses through aberrant expression of SRC-3 are responsible for uncontrolled
proliferation of human ATC [Figure 3B-IandII].

The fact that SRC-3 was both highly elevated and associated with increased proliferation provided the basis
to test the efficacy of SI-2 in the treatment of ATC . SI-2 treatment of cultured human ATC cell lines
[147]
(THJ-11T and -16T) markedly suppressed tumor cell proliferation by inducing apoptosis and impeding cell
cycle progression. Remarkably, growth of tumors derived from THJ-11T [Figure 4A-I] or -16T [Figure 4A-
II] cells was inhibited by SI-2. The mean tumor weight was reduced by 76% and 70%, respectively, in the SI-
2-treated group, compared to the vehicle-treated control group [Figure 4B-I and -II]. The inhibition of
tumor growth by SI-2 was due to induction of apoptosis as evidenced by the detection of high levels of
cleaved caspase 3 and pro-apoptotic regulators such as Bim in the xenograft tumors. In addition,
proliferation of tumor cells was reduced as evidenced by reduced levels of Ki-67 and cyclin D1. Moreover,
SI-2 blocked the activity of CSCs through inhibition of aldehyde dehydrogenase activity and expression.
This observation suggested that a global transcriptional program through SRC-3 is critical for maintaining
CSCs in ATC.

Finally, in-depth gene set enrichment analysis (GSEA) using The Cancer Genome Atlas Program-Thyroid
Cancer (TCGA-THCA) data confirmed extensive involvement of SRC-3 in the activation of multiple
oncogenic signaling pathways. The coordinated activation of 48 cancer-driver genes through SRC-3 signals
poor clinical outcome in human thyroid cancer. The GSEA further indicated that this involvement of SRC-3
occurred through enrichment of genetic regions occupied by oncogenic transcription factors such as the
MYC/MAX complex, NF-κB, E2F1, and ETS1 . These findings suggest that many different oncogenic
[147]
signaling pathways driven by multiple upstream driver mutations assembled on the transcription responses.
SRC-3 would have a critical role in the final manifestation of oncogenic transcription responses, and
therefore the identification of small-molecule inhibitors such as SI-2 to target SRC-3 is a promising strategy
for effective ATC treatment.

Transcription-associated cyclin-dependent kinases
The cyclin-dependent kinases (CDKs) are the families of serine/threonine kinases that mediate fundamental
cellular processes such as cell proliferation and survival. They can generally be classified into two major
groups: cell cycle-related CDKs (CDK1, CDK2, CDK4, and CDK6) and transcription-associated CDKs
(CDK7, CDK8, CDK9, CDK12, and CDK13). Each CDK is bound to a specific cyclin partner that guides the
CDK activity. Because of their important role in cancer cell survival and growth, they have been regarded as
promising therapeutic targets. Recently, CDK4/6 inhibitors have been shown to be effective in preclinical
studies of multiple cancer types [148-150] , and impressive clinical outcomes have been demonstrated in