Targeting CDK12-mediated transcription regulation in anaplastic
thyroid carcinoma
Meijuan Geng, Yiyi Yang, Xinyi Cao, Lin Dang, Tianye Zhang, Lirong Zhang*
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China
article info
Article history:
Received 13 September 2019
Accepted 4 October 2019
Available online xxx
Keywords:
Anaplastic thyroid carcinoma
CDK12
THZ531
Ser2 phosphorylation of pol II CTD
Chemoresistance
Super enhancer
abstract
Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid cancer, with no effective
treatment available. Identification of new anti-ATC drugs represents an urgent need. In this study, we
find that ATC cells are highly sensitive to THZ531, a potent inhibitor of the transcriptional cyclindependent kinase (CDK), CDK12. Cell-based assays demonstrate that CDK12 inhibition significantly
impedes cell cycle progression, induces apoptotic cell death, and impairs colony formation in ATC cells.
THZ531 causes a loss of elongating RNA polymerase II and suppresses gene expression in ATC cells. An
integrative analysis of gene expression profiles and super-enhancer landscape, combining with functional assays, leads to the discovery of two new ATC cancer genes, ZC3H4 and NEMP1. Furthermore,
CDK12 inhibition enhances the sensitivity of ATC cells to doxorubicin-mediated chemotherapy. Thus,
these findings indicate that CDK12 is a potential therapeutic target for ATC treatment and its inhibition
may help to overcome the chemoresistance in patients with ATC.
© 2019 Elsevier Inc. All rights reserved.
1. Introduction
Thyroid cancer is the most common malignancy in the endocrine system, and its incidence is increasing in recent decades. ATC
is the most aggressive subtype of thyroid cancer, which arises from
follicular thyroid cells. ATC is rare, representing only 1%e2% of
thyroid carcinomas, but accounts for nearly half of deaths from
thyroid cancer [1]. The clinical course of ATC is characterized by
rapidly growing thyroid mass with compressive symptoms and
high rates of metastasis [2]. Despite multimodal approaches
combining surgery, radiotherapy and/or chemotherapy, ATC is
associated with a poor prognosis, with a median overall survival of
only five months [3]. Therefore, there is an urgent need for developing novel effective treatments for ATC.
Cyclin-dependent kinases (CDKs) play critical roles in regulating
various cellular processes. The CDKs can be divided into two
functionally different subfamilies: the cell-cycle-related CDKs
(CDK1, 2, 4, and 6) and the transcription-related CDKs (CDK7, 8, 9,
11, 12, and 13) [4,5]. The transcriptional CDKs regulate gene
transcription by phosphorylating the C-terminal domain (CTD) of
RNA polymerase II (Pol II) as well as various transcription regulating
factors [5].
CDK12 is a transcription-related CDK that complexes with cyclin
K to regulate gene transcription [6]. CDK12 has been demonstrated
to be essential for DNA damage response (DDR), mRNA processing,
and differentiation [7e9]. Several recent studies also implicated
CDK12 in cancer pathology [10,11]. Genomic mutations in CDK12,
as well as its overexpression, have been detected in various cancers,
including breast, ovarian, oesophageal, stomach, endometrial,
uterine, bladder, colorectal, and pancreatic cancers [10]. THZ531 is a
covalent inhibitor of CDK12, which shows strong anti-tumor effects
in several cancers by inhibiting CDK12 kinase activity [12,13]. In this
study, we aim to investigate the roles of CDK12 in ATC pathology
and the therapeutic potential of CDK12 inhibition in ATC.
2. Materials and methods
2.1. Cell culture conditions
CAL-62 and 8505C cell lines were provided as a generous gift
from Professor Haixia Guan (The First Hospital of China Medical
University, P.R. China). C643 cell line was purchased from the Chinese Academy of Science (Shanghai, P.R. China). CAL-62 cells were
cultured in Dulbecco’s Modified Eagle Medium supplemented with
Abbreviations: CDK, cyclin-dependent kinase; ATC, anaplastic thyroid carcinoma; Pol II, polymerase II; CTD, C-terminal domain; pSer2, phosphorylation of
Serine 2; DOX, doxorubicin.
* Corresponding author.
E-mail address: [email protected] (L. Zhang).
Contents lists available at ScienceDirect
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10% fetal bovine serum (FBS). 8505C and C643 cells were cultured
in RPMI 1640 medium þ10% FBS þ1% penicillin/streptomycin.
2.2. Cell proliferation assay
Cells were seeded into 96-well plates at a density of 2, 000 cells
per well in triplicate and cultured overnight. Cells were treated
with drugs for 48 h, then cell viability was measured using the
CellTiter96® AQueous One Solution Cell Proliferation Assay (MTS)
(Promega cat# G3581) kit. THZ531 and doxorubicin hydrochloride
were purchased from MedChemExpress (Monmouth Junction, NJ).
2.3. Cell-cycle and apoptosis analysis
Cells were seeded into 6-well plates and cultured overnight.
Cells were treated with THZ531 for the indicated time. For cell cycle
analysis, cells were fixed with 80% ethanol at 4 C overnight, followed by resuspension with PI/RNase Staining Buffer solution. Cell
apoptosis analysis was performed using the FITC Annexin V
Apoptosis Detection Kit I (BD Biosciences) following the manufacturer’s protocol.
Cell cycle and apoptosis rate were measured using BD FACS
Verse (Becton Dickinson, Franklin Lakes, NJ).
2.4. Colony formation assay
Cells were seeded into 6-well plates at a concentration of
200 cells/well. Cells were fed with growth media for 10 days, followed by staining with crystal violet solution.
2.5. Western blotting
Whole-cell lysates were extracted with RIPA buffer supplemented with EDTA free Protease Inhibitor Cocktail (Roche) and
PhosSTOP Phosphatase Inhibitor (Roche). Protein concentrations
were measured by the Bradford protein assay. Equal amounts of
protein samples were separated by SDS-PAGE and transferred to
nitrocellulose membranes, followed by immunoblotting with primary antibodies against pSer2 Pol II (Millipore Cat. No. 04-1571),
pSer5 Pol II (Millipore Cat. No. 04-1572), pSer7 Pol II (Millipore Cat.
No. 04-1570), Pol II (Abcam Cat. No. ab817); CDK12 (Cell Signaling
Technology, Cat. No.11973), PARP (Cell Signaling Technology Cat.
No. 9542) and a-tubulin (Proteintech Cat. No.11224-1-AP).
2.6. Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde for 15 min at RT,
permeabilized with 1% Triton X-100 for 5 min, then blocked with
5% BSA in PBS for 1 h at RT. Slides were incubated with primary
antibody against gH2AX (Ser139) (Millipore Cat. No.05-636), followed by FITC conjugated secondary antibodies. Slides were
mounted using DAPI reagent (Sigma Cat. No.6057). Three hundred
cells per sample were counted. A cell with >5 foci per nucleus was
counted as gH2AX positive [13].
2.7. RNA-seq analysis and qRT-PCR validation
CAL-62 cells were treated in duplicate with either DMSO or
THZ531 for 6 h, and then total RNA was collected using TRIzol reagent (Thermo Fisher Cat. No.15596018). Each RNA sample was
subjected to Oligo-dT and adaptor ligation. Sequencing libraries
were sequenced on NovaSeq sequencer (Illumina). Clean reads
were mapped to the human hg19 reference genome. Differentially
expressed genes were identified using DESeq2 algorithms. Gene
ontology (GO) analysis was performed with the DAVID 6.8 online
tools. Quantitative reverse transcriptase-polymerase chain reaction
(qRT-PCR) was performed using Transcriptor cDNA synthesis Kit
(Roche Cat. No.4897030001) and the FS Universal SYBR Green
Master Mix (Roche, 4913914001), run in 96-well format on the
7500 FAST Real-time PCR system (Applied Biosystems), according
to the manufacturer’s protocol. The primers used for qRT-PCR
validation are shown in Supplementary Table 1.
2.8. ChIP-seq analysis
CAL-62 cells were fixed with 1% formaldehyde for 10 min at
room temperature (RT). Cross-linking was stopped with 150 mM
glycine. Cells were harvested and sonicated to obtain chromatin
fragments (100e500 bp) with SONICS Vibra-cell™. Immunoprecipitation was performed with RNAPII CTD pSer2 antibody (Cell
Signaling Technology, Cat. No.13499). Purified DNA was sequenced
on X-ten sequencer (Illumina). ChIP-seq clean reads were mapped
to the human hg19 reference genome using Bowtie2. ChIP-seq
peaks were identified with MACS2 as previously described [14].
2.9. Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.
Unpaired two-tailed Student’s t-test was used to calculate p-value
between two groups of independent samples, one-way analysis of
variance (ANOVA) was used to compare multiple groups.
3. Results
3.1. CDK12 inhibition impairs ATC growth
Our previous screening in CAL-62 ATC cells identified THZ531 as
a potent inhibitor of cell growth [15]. To confirm this anti-ATC activity of THZ531, we further evaluate the growth-inhibitory effect of
THZ531 in three representative ATC cell lines (CAL-62, 8505C, and
C643). Consistently, THZ531 dose-dependently reduced cell growth
in all ATC cell lines tested (Fig. 1A). Moreover, ATC cells treated with
THZ531 underwent marked cell-cycle arrest in the G2/M stage
(Fig. 1B). We next assessed whether THZ531 treatment caused
apoptotic cell death in ATC cells. THZ531 increased the Annexin V
and propidium iodide positivity (Fig. 1C), and the increase of PARP
cleavage was also observed in cells treated with THZ531 (Fig. 1D),
supporting that THZ531 induces apoptotic cell death in ATC cancer
cells. Colony formation experiments demonstrated that THZ531
strongly inhibited the colony formation of ATC cells (Fig. 1E).
Notably, depletion of CDK12 expression by CRISPR/cas9-mediated
gene editing (Fig. 1F) also impaired the cell growth (Fig. 1G) and
reduced colony formation (Fig. 1H) in ATC cells, suggesting that
CDK12 is the pharmacological target of THZ531 in ATC cells.
3.2. THZ531 inhibits transcriptional elongation in ATC cells
We next sought to understand the molecular mechanisms underlying the cytotoxic effects of THZ531 on ATC cells. Studies have
shown that CDK12 phosphorylates the elongation-associated
serine 2 (Ser2) of the RNA Pol II CTD, thus promoting the transcriptional elongation of regulated genes [6,7]. To assess whether
CDK12 kinase activity is required for Pol II CTD phosphorylation in
ATC cells, we treated CAL-62 cells with escalating doses of THZ531
for 6 h. Indeed, as a covalent inhibitor of CDK12, THZ531 selectively
decreased Ser2 phosphorylation levels in a dose-dependent
manner in CAL-62 cells, with minimal effect on CTD pSer5 or
pSer7 levels (Fig. 2A).
To investigate how these findings extend to gene-specific pSer2
signal, we performed a genome-wide analysis of pSer2 signal by
2 M. Geng et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: M. Geng et al., Targeting CDK12-mediated transcription regulation in anaplastic thyroid carcinoma, Biochemical and
Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.052
chromatin immunoprecipitation coupled with sequencing
(ChIPeSeq). Consistent with the findings that pSer2 is an active
mark of elongating Pol II, the pSer2 ChIPeSeq profiles show pronounced pSer2-Pol II signal in the gene body, with a peak near the
30 end of genes (Fig. 2B). Notably, THZ531 treatment led to a substantial shift of pSer2 occupancy toward the transcriptional start
site (TSS) (Fig. 2B). The pSer2 ChIPeSeq profiles of two representative genes are shown in Fig. 2C. Thus, these results demonstrate
that THZ531 inhibits transcriptional elongation in ATC cells, which
may cause the reduction of gene expression.
3.3. CDK12 inhibition preferentially downregulates DNA damage
repair genes
To investigate the effect of THZ531 on the gene expression
profiles of ATC cells, we proceeded to perform whole-transcriptome
sequencing (RNA-Seq) analysis. Consistent with the fact that CDK12
is essential for the gene transcription elongation, CDK12 inhibition
by THZ531 treatment caused a profound reduction of gene
expression in ATC cells (Fig. 2D). We termed the group of genes
(whose expression was decreased over twofold after THZ531
treatment for 6 h) as “THZ531-sensitive genes”. Interestingly, a
considerable proportion of genes were upregulated by THZ531,
which was possibly induced by negative feedback or other stress
responses.
To analyze which cellular pathways were particularly sensitive
to CDK12 inhibition, we performed gene ontology (GO) analysis of
THZ531-sensitive genes. Notably, genes involved in DNA damage
repair were among the most sensitive to CDK12 inhibition in ATC
cells (Fig. 2E), suggesting that these genes likely mediate the potent
anti-ATC effect of THZ531.
3.4. Identification of candidate oncogenic genes in ATC
Recent studies have demonstrated that oncogenic genes are
frequently associated with super-enhancers (SEs) [16,17]. The SEFig. 1. Inhibition of CDK12 by THZ531 inhibits ATC cell growth. (A) Proliferation assay (MTS) showing the effects of THZ531 treatment in representative ATC cell lines at indicated
time points. (B) Cell cycle analysis of cells exposed to THZ531. Cells were treated with escalating doses of THZ531 for 48 h. Cell cycle distribution was measured by FACS analysis with
propidium iodide (PI) staining. (C) Apoptosis analysis in CAL-62 cells. CAL-62 cells were treated with THZ531 (100 nM) for 48 or 72 h. Cell apoptosis was measured by FACS analysis
with FITC-Annexin V and PI staining. (D) Immunoblotting analysis of PARP cleavage in CAL-62 cells exposed to THZ531 (200 nM). (E) Colony formation assay in CAL-62 and 8505C
cells exposed to THZ531 (100 nM). (FeH) CAL-62 cells were infected with lentivirus carrying Cas9 and sgCDK12 or control sgRNA. Expression of CDK12 was evaluated by Western
blot analysis (F). Cell viability was evaluated by MTS assay (G). Colony formation assay in CAL-62 cells with CDK12 depletion (H). Data represent mean ± SD of three replicates.
*P < 0.05, ***P < 0.001.
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associated oncogenes often exhibit high expression and are highly
sensitive to transcription inhibition [18e22]. Our previous study
characterized the SE landscape of ATC and revealed that some SEassociated genes play key roles in ATC pathogenesis [15]. To
further identify the key THZ531-sensitive oncogenic genes in ATC,
we performed an integrative analysis of the RNA-Seq profiles and
the SE profiles obtained from ATC cells. The candidate THZ531-
sensitive oncogenic genes were selected as (1) associated with
SEs, (2) highly sensitive to low-dose (100 nM) THZ531, and (3)
expression levels within the top 20% of all active transcripts. As a
result, 12 candidate oncogenic genes were identified (Fig. 3A). The
ChIP-Seq profiles of two representative genes, ZC3H4 and LEMD2,
are shown in Fig. 3B. The RNA-Seq signals of these candidate genes
are shown in Fig. 3C. The suppression of these candidates by
THZ531 was further validated by qRT-PCR analysis (Fig. 3D). Three
of these candidates have been reported to play important roles in
cancer, including TNFRSF1A [23], NEMP1 [24], and SPC24 [25e27].
Among them, SPC24 had been reported to play critical roles in ATC
initiation and progression [26]. Our previous study also identified
SMG9 and TNFRSF1A as SE-associated genes that are sensitive to
THZ1, a potent inhibitor of CDK7. Depletion of these two genes
impairs the colony formation of ATC cells [15]. Notably, this integrative analysis also identified nine new candidates whose roles in
ATC have not been reported before. To investigate the roles of these
new candidate oncogenic genes in ATC, all of them were silenced
using CRISPR/Cas9-mediated gene editing in ATC cells, and the
colony formation ability was assessed after gene depletion. As
shown in Fig. 3E, depletion of five candidate genes significantly
impaired the colony formation in ATC cells. Silencing of ZC3H4 and
NEMP1 caused the most significant reduction of colony formation
(Fig. 3E).
Fig. 2. THZ531 inhibits transcriptional elongation in ATC cells. (A) Immunoblotting analysis of the phosphorylation of RNA Pol II in CAL-62 cells treated with DMSO or THZ531 for
6 h. (B) Metagene analysis of global occupancy of Ser2-phosphorylated (pSer2) Pol II. TSS, transcriptional start site; TTS, transcriptional termination site. (C) ChIP-Seq tracks of two
representative THZ531-sensitive genes. (D) Heatmap of gene expression values in CAL-62 cells treated with DMSO or THZ531 for 6 h. Rows show Z scores calculated for each dose of
THZ531 versus DMSO. (E) Gene ontology analysis of the THZ531-sensitive genes in CAL-62 cells.
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Please cite this article as: M. Geng et al., Targeting CDK12-mediated transcription regulation in anaplastic thyroid carcinoma, Biochemical and
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3.5. THZ531 sensitizes the ATC cells to conventional chemotherapy
Doxorubicin (DOX) is the first-line drug in the treatment of ATC
[1]. However, the single-agent chemotherapy using DOX alone
achieves a poor response rate (only 20%) in patients with advanced
ATC [28]. Thus, searching for therapeutic combinations to improve
the outcome of chemotherapy is urgently needed. We observed
that THZ531 effectively inhibited ATC cell growth. DOX is known to
be a DNA-damaging agent. Our RNA-Seq data suggest that THZ531
preferentially represses the DNA damage repair genes in ATC cells.
Thus, we sought to determine if combing DOX with THZ531 could
achieve better anti-ATC effects than using DOX alone. We first
investigated the effects of the combination of THZ531 and DOX on
DNA damage and repair by performing immunofluorescent analysis
of gH2AX, an indicator of DNA damage. As shown in Fig. 4A and B,
THZ531 or DOX alone induced gH2AX foci staining in CAL-62 cells,
and the combination of these two drugs significantly increased the
formation of gH2AX foci, suggesting that this combination impairs
the ability of ATC cells to repair DNA damage. We next investigated
whether THZ531 could enhance the DOX sensitivity in ATC cells. As
shown in Fig. 4C, THZ531 strongly enhanced the anti-growth effect
of DOX in ATC cells. Taken together, these results indicate that inhibition of CDK12 by THZ531 enhances the potency of conventional
chemotherapy in ATC cells.
4. Discussion
ATC is the most aggressive thyroid cancer and lacks effective
treatments. Nowadays, molecular-based targeted therapies have
been proved to be the most important strategies to treat many
aggressive cancers, such as non-small cell lung cancers with EGFR
or ALK mutations, for which therapeutics designed to target EGFR
or ALK mutations have been shown to be extremely effective [29].
Recent large-scale genome analyses of ATCs revealed that ATCs had
a greater mutational burden compared to other types of cancer
[30e32]. The high genetic complexity of ATCs makes it challenging
to dissect the molecular mechanisms of ATC pathogenesis, and
therefore impeding the development of effective targeted therapies
for patients with ATC. Here, we demonstrate that a CDK12 inhibitor,
THZ531, strongly inhibits ATC cell growth and induces apoptosis.
These findings suggest that targeting transcriptional processes, as
opposed to specific genomic mutations, might provide therapeutic
value against ATCs.
The molecular mechanisms underlying ATC progression are
poorly defined. This study identified 12 candidate cancer genes in
ATC by integrative analysis of gene expression profiles and SE
profiles. One of the candidates, SPC24, has been reported to play
critical roles in ATC progression [26]. Furthermore, this integrative
analysis also identified several candidate oncogenic genes, two of
them (SMG9 and TNFRSF1A) were also identified as candidate
Fig. 3. Identification of candidate oncogenic genes in ATC. (A) Venn diagram showing overlap of THZ531-sensitive genes (THZ531 100 nM), SE-associated genes, and top
expressed genes. (B) H3K27ac ChIP-Seq profiles of two representative SE-associated genes. (C) Heatmap showing the RNA-Seq signals of candidate oncogenic genes identified in (A).
(D) qRT-PCR validation of repression of candidate oncogenic genes by THZ531 treatment (100 nM, 6 h). (E) Colony formation assay of CAL-62 cells depleted with indicated genes.
Data represent mean ± SD of three replicates. **P < 0.01, ***P < 0.001.
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Please cite this article as: M. Geng et al., Targeting CDK12-mediated transcription regulation in anaplastic thyroid carcinoma, Biochemical and
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oncogenes in our previous study [15]. SMG9, ZC3H4, and NEMP1,
whose depletion resulted in more than 50% of reduction of colony
formation in ATC cells, suggesting these genes might be critical for
ATC cell growth, which requires further investigation in future.
In summary, the current study demonstrates that targeting
transcriptional elongation by CDK12 inhibition is a promising
therapeutic strategy for ATC treatment. Moreover, through the
integrative analysis of the gene expression profiles and SE landscape of ATC, this work identifies several candidate oncogenic
genes of ATC, thus offering insights into the molecular mechanisms
underlying ATC pathogenesis. Furthermore, our data also indicate
that inhibition of CDK12 by THZ531 may help to overcome the
chemoresistance in ATC.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant No. 31571336).
Transparency document
Transparency document related to this article can be found
online at http://doi:10.1016/j.bbrc.2019.10.052
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.10.052.
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M. Geng et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 7
Please cite this article as: M. Geng et al., Targeting CDK12-mediated transcription regulation in anaplastic thyroid carcinoma, Biochemical and
Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.052