Synthesis and mechanistic studies of quinolin-chlorobenzothioate derivatives with proteasome inhibitory activity in pancreatic cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2018.09.037

Inhibition of proteasome activity blocks the degradation of dysfunctional proteins and induces cancer cell death due to cellular stress. Thus, proteasome inhibitors represent an attractive class of anticancer agents, and bortezomib, carfilzomib and ixazomib have been FDA-approved to treat multiple myeloma. However, cancer cells acquire resistance to these inhibitors through point mutations in the proteasome catalytic subunit or induction of alternative compensatory mechanisms. In this study, we identified a quinolin-chlorobenzothioate, QCBT7, as a new proteasome inhibitor showing cytotoxicity in a panel of cancer cell lines. QCBT7 is a more stable derivative of quinoline-8-thiol that targets the regulatory subunit instead of the catalytic subunit of the proteasome. QCBT7 caused the accumulation of ubiquitylated proteins in the cancer cells, indicating its proteasome inhibitory activity. Additionally, QCBT7 increased the expression of a set of genes (PFKFB4, CHOP, HMOX1 and SLC7A11) at both nascent RNA and protein levels, similarly to the known proteasome inhibitors MG132 and ixazomib. Together, QCBT7 induces proteasome inhibition, hypoxic response, endoplasmic reticulum stress and glycolysis, finally leading to cell death. Importantly, we have identified PFKFB4 as a potential biomarker of proteasome inhibitors that can be used to monitor treatment response.

Full text of DOI:10.1016/j.ejmech.2018.09.037

Accepted Manuscript
Synthesis and mechanistic studies of quinolin-chlorobenzothioate derivatives with
proteasome inhibitory activity in pancreatic cancer cell lines
Shuai Hu, Yi Jin, Yanghan Liu, Mats Ljungman, Nouri Neamati
PII:
S0223-5234(18)30809-2
10.1016/j.ejmech.2018.09.037
EJMECH 10748
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2018
Revised Date: 14 September 2018
Accepted Date: 14 September 2018
Please cite this article as: S. Hu, Y. Jin, Y. Liu, M. Ljungman, N. Neamati, Synthesis and mechanistic
studies of quinolin-chlorobenzothioate derivatives with proteasome inhibitory activity in pancreatic
cancer cell lines, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.037.
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Synthesis
quinolin-chlorobenzothioate derivatives
inhibitory activity in pancreatic cancer cell lines
and
mechanistic
studies
of
with proteasome
Shuai Hu a, b, Yi Jin a, c, *, Yanghan Liu a, Mats Ljungman d, Nouri Neamati a, *
a Department of Medicinal Chemistry, College of Pharmacy, Rogel Cancer Center,
University of Michigan, Ann Arbor, Michigan 48109, United States
b Department of Computational Medicine and Bioinformatics, University of
Michigan, Ann Arbor, Michigan 48109, United States
c Key Laboratory of Medicinal Chemistry for Natural Resource, School of
Chemical Science and Technology, Yunnan University, Kunming 650091, China
d Department of Radiation Oncology, Rogel Cancer Center, University of Michigan,
Ann Arbor, Michigan 48109, United States
Corresponding authors: Nouri Neamati, Yi Jin
Corresponding author email: neamati@umich.edu

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Abstract
Inhibition of proteasome activity blocks the degradation of dysfunctional proteins
and induces cancer cell death due to cellular stress. Thus, proteasome inhibitors
represent an attractive class of anticancer agents, and bortezomib, carfilzomib and
ixazomib have been FDA-approved to treat multiple myeloma. However, cancer
cells acquire resistance to these inhibitors through point mutations in the
proteasome catalytic subunit or induction of alternative compensatory
mechanisms. In this study, we identified a quinolin-chlorobenzothioate, QCBT7, as
a new proteasome inhibitor showing cytotoxicity in a panel of cancer cell lines.
QCBT7 is a more stable derivative of quinoline-8-thiol that targets the regulatory
subunit instead of the catalytic subunit of the proteasome. QCBT7 caused the
accumulation of ubiquitylated proteins in the cancer cells, indicating its
proteasome inhibitory activity. Additionally, QCBT7 increased the expression of a
set of genes (PFKFB4, CHOP, HMOX1 and SLC7A11) at both nascent RNA and
protein levels, similarly to the known proteasome inhibitors MG132 and ixazomib.
Together, QCBT7 induces proteasome inhibition, hypoxic response, endoplasmic
reticulum stress and glycolysis, finally leading to cell death. Importantly, we have
identified PFKFB4 as a potential biomarker of proteasome inhibitors that can be
used to monitor treatment response.
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Keywords
Quinolin-chlorobenzothioate; Proteasome inhibitor; Hypoxia; Endoplasmic
reticulum stress; Glycolysis; Pancreatic cancer
1. Introduction
The human 26S proteasome is an essential protein degradation machinery
designated to maintain cellular homeostasis via removal of unfolded and
misfolded proteins, and it is involved in balancing cell survival and apoptotic cell
death. Importantly, small molecule proteasome inhibitors activate unfolded protein
response leading to cancer cell death and are attractive for cancer therapy [1, 2].
Bortezomib, ixazomib and carfilzomib were approved for treating multiple
myeloma or mantle-cell lymphoma as proteasome inhibitors. They have boronic
acid or epoxyketone scaffold, known to interact with the N-terminal threonine
residues in the catalytic subunits of the proteasome thus blocking its activity
(Figure 1) [1]. Derivatives of the approved proteasome inhibitors have shown
efficacy in multiple cancers and are currently undergoing clinical trials in solid
tumors, including pancreatic cancer [1-4]. Unfortunately, patients frequently
develop resistance to these therapies due to either point mutations on the drug
binding site or induction of alternative compensatory mechanisms such as the
aggresome pathway [5]. Therefore, development of proteasome inhibitors with
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different scaffolds and mechanisms of action would be critical to overcome tumor
cell resistance to first-line proteasome inhibitors.
Pancreatic cancer is a difficult cancer to treat due to its rapid metastatic
spread and late-stage diagnosis [6]. Pancreatic ductal adenocarcinoma (PDAC) is
the major type of pancreatic cancer and is predicted to become the second most
common cause of cancer-related death within the next decade in the United
States [7, 8]. Pancreatic tumor cells can rapidly develop resistance to current
chemotherapies, such as gemcitabine, nab-paclitaxel and FOLFIRINOX [9, 10].
Thus, it is essential to develop more effective therapeutics to better treat this
dreadful disease. Inhibition of the ubiquitin-proteasome pathway contributes to
apoptotic cell death in pancreatic cancer [11, 12]. Bortezomib sensitizes
pancreatic cancer cells to endoplasmic reticulum (ER) stress and apoptosis [13].
Additionally, proteasome activator subunit 3 (PSME3) promotes the pancreatic
cancer growth by activating the proteasome activity [14]. Therefore, the
proteasome is an attractive target for therapeutics in pancreatic cancer.
In a medium-throughput phenotypic screen of 20,000 diverse drug-like
compounds, we identified
a
quinolin-chlorobenzothioate, QCBT7, with
submicromolar cytotoxicity in the colon carcinoma cell line HCT 116. It was
previously shown that the structurally similar compound 8-quinolinethiol
hydrochloride (8TQ) inhibits the essential proteasome deubiquitinase Rpn11,
instead of the catalytic subunit of the proteasome, suggesting that QCBT7 may
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also affect proteasome function (Figure 1) [15]. Quinoline derivatives possess
antibacterial, antimalarial and anticancer activities. Thioester, amide and ester
derivatives of quinoline have been used in antimicrobial and anticancer research
[16-19]. To further identify new quinoline derivatives with potential proteasome
inhibition and anticancer activities, we synthesized 21 analogs of QCBT7 to
establish structural requirement for potency. We then profiled nascent RNA and
protein expression in QCBT7-treated pancreatic cancer cells to understand how
these cells respond to the treatment. Our results demonstrate that the induced
transcription and protein patterns following QCBT7 treatment resemble those
obtained for MG132 and ixazomib, indicating that QCBT7 blocks proteasome
activity and induces hypoxic response, ER stress and glycolysis.
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Figure 1. Structures of select proteasome inhibitors and the lead compound
QCBT7. Carfilzomib and epoxomicin are epoxyketone-based compounds, and
bortezomib and ixazomib are boronate-containing compounds. MG132 is a
peptide aldehyde. 8TQ is 8-quinolinethiol hydrochloride. QCBT7 has a similar
scaffold to 8TQ.
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2. Results and discussion
2.1. Identification of QCBT7
In a medium-throughput screen of 20,000 drug-like compounds derived from a
diverse library, QCBT7 was one of the most cytotoxic hits and had an IC50 value of
0.6 µM in HCT 116 cells [20]. QCBT7 was further tested in the pancreatic cancer
cell lines, showing IC50 values of 2.6 µM and 1.1 µM in MIA PaCa-2 and PANC-1
cells, respectively (Table 1). Considering the stability of the thioesters in the
cellular reducing environment, we synthesized ester and amide derivatives of
QCBT7 to potentially increase the stability and maintain cytotoxicity.
2.2. Synthesis of QCBT7 and its derivatives
Derivatives of QCBT7 were prepared according to the general procedure shown
in Scheme 1. Benzoyl chloride or benzyl chloride was reacted with 8-quinolinethiol
hydrochloride (8TQ), 8-hydroxylquinoline or 8-aminoquinoline, respectively, in the
presence of potassium carbonate in dimethylformamide (DMF) to afford final
products 3a-3u in 72-95% yields (Supplementary Figure 1S).
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Scheme 1. Preparation of QCBT7 derivatives.
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2.3. 8-thioester and 8-ester quinoline derivatives are more cytotoxic than
8-amide quinoline derivatives
Cytotoxicity of QCBT7 and its derivatives was assessed using the colorimetric
MTT assay in a panel of cancer cell lines (Table 1). The 8-thioester derivatives
(3a-d, 3f, 3g) had similar IC50 values around 1 - 4
µM，while the 8-ester derivatives
(3h-k, 3m, 3n) were less potent (IC50: 5-10 M). Different substituents on the
µ
benzene ring (R group in Scheme 1) had little effect on the cytotoxicity of the
compounds. The 8-amide derivatives (3o-r, 3t, 3u) were much less cytotoxic (IC50
> 20 µM) although the amide-quinoline was more stable than the ester-quinoline
and thioester-quinoline in the cellular reducing environment. The ether analogs 3e
and 3l were inactive. These results suggest that the thioester-quinoline and
ester-quinoline scaffolds are responsible for the cytotoxicity of this series of
compounds. Due to the substantial reduction in cytotoxicity of the amide-quinoline
derivatives and similar high potency among 8-thioeter-quinoline analogs, we
selected QCBT7 for further stability and mechanistic studies.
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Table 1. Cytotoxicity of 8-thioester/ester/amide-quinoline derivatives of QCBT7 in
a panel of cancer cell lines.
IC50 (µM)
Compound
MIA PaCa-2
PANC-1
HCT 116
KYSE-70
3a
1.1 ± 0.3
1.3 ± 0.1
0.8 ± 0.1
0.7 ± 0.1
>20
3.3 ± 0.2
3.4 ± 0.3
2.6 ± 0.6
2.4 ± 0.8
>20
1.5 ± 0.4
1.5 ± 0.6
1.1 ± 0.1
0.9 ± 0.3
>20
1.1 ± 0.4
1.1 ± 0.6
0.6 ± 0.3
0.5 ± 0.3
>20
3b
3c (QCBT7)
3d
3e
3f
0.9 ± 0.2
0.6 ± 0.1
11.8 ± 0.5
6.9 ± 0.1
15.2 ± 5.7
6.6 ± 0.2
>20
4.9 ± 3.3
3.6 ± 0.1
10.3 ± 5.6
6.5 ± 0.7
6.5 ± 0.1
5.4 ± 0.4
>20
1.7 ± 0.1
0.9 ± 0.4
9.4 ± 4.4
5.1 ± 2.1
7.1 ± 1.1
4.9 ± 1.4
>20
1.5 ± 0.6
0.7 ± 0.1
4.7 ± 1.5
4.0 ± 0.7
3.0 ± 0.8
3.7 ± 1.1
>20
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4.2 ± 2.0
2.9 ± 0.1
>20
4.1 ± 0.3
6.5 ± 0.3
>20
7.0 ± 0.4
5.8 ± 0.8
>20
7.6 ± 1.2
5.5 ± 0.2
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
3s
3t
>20
>20
>20
>20
>20
>20
>20
>20
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3u
>20
>20
>20
>20
Data are shown as mean ± standard deviation from at least three independent
experiments.
2.4. QCBT7 is more stable than 8TQ in solution
To assess the chemical stability of QCBT7, we performed liquid chromatography
mass spectrometry (LCMS) of QCBT7 at 3 different time points after it was added
to DMSO or to a 1:1 DMSO/water solution. N-acetylcysteine (NAC) was added in
the solution to simulate a reducing environment and react with free thiols in the
solution. 8TQ is the parent compound from which QCBT7 was synthesized, and is
likely to be the functional scaffold for this series of compounds. QCBT7 was more
stable than 8TQ under a range of conditions (Table 2, Supplementary Figure 2S).
In 100% DMSO, QCBT7 was still intact after 7 days, while 8TQ formed dimers
once it was dissolved in DMSO. Addition of NAC did not affect the results, and
8TQ also reacted with NAC forming a conjugate at 2 and 5 hours. In a 1:1 solution
of DMSO and water, 8TQ formed dimers within one minute, while QCBT7 was
stable and present in a monomeric form after 48 hours. Additionally, NAC reacted
with 8TQ as early as one minute, but did not react with QCBT7 in the 1:1 solution.
In conclusion, QCBT7 is more stable than 8TQ in DMSO and 1:1 solution of
DMSO and water, and NAC has little effect on QCBT7 even after 48-hour
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incubation. Therefore, we selected QCBT7, the more stable compound, for
mechanistic studies.
Table 2. Stability of (a) QCBT7 and (b) 8TQ in DMSO and 1:1 DMSO/water
solution with or without NAC.
(a) Percent of QCBT7 remaining
Time / h
DMSO
N/A
DMSO+NAC 1:1 DMSO/water 1:1 DMSO/water+NAC
2
100
100
100
100
100
100
24
N/A
100 (> 7
days)
48
97.8
97.6
100
(b) Percent of 8TQ remaining
Time / h
0.017
0.5
DMSO
74.9
DMSO+NAC
72.8
1:1 DMSO/water
1:1 DMSO/water+NAC
78.7
N/A
72.2
N/A
34.6
N/A
2
5
0
13.1
0
13.3
0
7.6
0
N/A
N/A: not measured at the time point. LCMS spectrums are shown in the
Supplementary Figure 2S.
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2.5. QCBT7 upregulates gene sets related to proteasome inhibition, UPR,
glycolysis and hypoxia
To better elucidate the potential mechanisms of action of QCBT7 in pancreatic
cancer, we utilized Bru-seq technique to assess its effects on nascent
transcription signatures in MIA PaCa-2 cells. After cells treated with 3.3 µM
QCBT7 for 4 h, we observed 326 upregulated genes with fold change (FC) over 2
and 127 downregulated genes with FC below 0.5 (Table 3, Supplementary Figure
3S). In the set of upregulated genes, STRING analysis revealed three major
biological processes: carbon metabolism, HIF-1 signaling pathway and glycolysis
[21]. We performed gene set enrichment analysis (GSEA) using all the genes
ranked by the log2-fold change from the Bru-seq results [22, 23]. The
CONCANNON_APOPTOSIS_BY_EPOXOMICIN_UP gene set was ranked as the
13th enriched gene set of the C2 curated gene sets from the Molecular Signatures
Database (MSigDB), suggesting that QCBT7 causes a similar transcriptional
response to proteasome inhibitor epoxomicin (Figure 2A, Supplementary Table
1S). Epoxomicin is a potent, selective, irreversible and cell-permeable 20S
proteasome inhibitor (Figure 1) [24]. Additionally, we observed the enrichment of
genes involved in the unfolded protein response (UPR), glycolysis and hypoxia in
the Hallmark gene sets (Figure 2B-D). Notably, a much higher number of
hypoxia-related genes were upregulated (158) than downregulated (13) by
QCBT7 (Figure 2E). Other gene sets indicated that QCBT7 also stimulated the
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transcription of genes involved in apoptotic cell death. Taken together, the results
from STRING and GSEA are consistent, and we hypothesize that QCBT7 causes
proteasome inhibition, hypoxic response, glycolysis and unfolded protein
response.
Figure 2. QCBT7 upregulated the gene sets related to proteasome inhibition,
unfolded protein response (UPR), glycolysis and hypoxic response. (A-D) GSEA
enrichment plots for top enriched gene sets matched with upregulated genes from
QCBT7 Bru-seq sample (FDR <= 0.001). (A) Epoxomicin, a proteasome inhibitor.
(B) UPR. (C) Glycolysis. (D) Hypoxia. Size is the number of genes, and NES is the
normalized enrichment score for each gene set. (E) Log2FoldChange of 1387
hypoxia-related genes from QCBT7 Bru-seq results. 1 and -1 are used as the
cutoff values for upregulated and down-regulated genes.
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Table 3. Top 50 upregulated genes in QCBT7-treated MIA PaCa-2 cells.
Gene
FC
Gene
FC
Gene
FC Gene FC Gene FC
HMOX1
149
BNIP3L
ADM
15
14
13
BTG1
PGK1
IL8
9
9
9
8
8
8
8
8
8
8
ENO2
GLA
8
7
7
7
7
6
6
6
6
6
ZNF395
GBE1
PAM
6
6
6
6
6
5
5
5
5
5
LOC344887 45
OSGIN1
PFKFB4
MIR210HG
SLC7A11
DOK3
36
32
30
27
24
23
21
19
GCLC
PDK3
TXNRD1
EID3
12 ANGPTL4
HK2
TNFSF9
12
NQO1
EGLN1
DDIT4
SRXN1
P4HA1
JUNB
PIR
PGD
FAM162A 12
PDK1 11
INSIG2
DUSP5
CCNG2
RNF24
ERO1L
SCAND2
ANKRD37
P4HA2
ARRDC3 10 DNAJB4
RNF122
CHAC1
9
9
HILPDA
C3orf58
UPRT
WSB1
FC is fold change, defined as treatment over control. HMOX1
PGK1 EGLN1 ENO2 and HK2 are involved in HIF-1 signaling pathway. PGK1
ENO2 HK2 and PGD are related to carbon metabolism. PGK1 ENO2 and HK2
affect glycolysis.
, PFKFB4, PDK1,
,
,
,
,
,
2.6. QCBT7 has similar expression signatures to proteasome inhibitors and
hypoxia inducers
To further test our hypothesis that QCBT7 inhibits proteasome activity and
induces hypoxia, glycolysis and ER stress, we performed connectivity map
(CMAP) analysis of QCBT7 Bru-seq results (Table 4) [25]. In the top 50 correlated
perturbagens (small molecules and genetic reagents causing gene expression
changes in cell lines), knockdown of PSMD1, PSMB5 and PSMA1 are listed,
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which encode different proteasome subunits. Additionally, proteasome inhibitors,
such as MG132 and MLN-2238 (ixazomib), display similar expression signatures
to QCBT7. Hypoxia-inducible factor (HIF) activators, such as VU-0418947-2 and
VU-0418946-1, also show high similarity (high connectivity score), suggesting that
QCBT7 induces hypoxic response. Taken together, CMAP analysis supports the
hypothesis that QCBT7 causes proteasome inhibition and elicits a hypoxic
response.
Table 4. Connectivity map analysis of QCBT7. Top compound perturbagens (CP),
gene over-expression perturbagens (OE), gene knock-down perturbagens (KO)
and perturbational class member (PCL) are listed.
Type
Name
Description
Pc_selection
CP
VU-0418947-2
hypoxia inducible factor activator
99.89
CP
CP
CP
OE
KD
VU-0418946-1
MG-132
hypoxia inducible factor activator
proteasome inhibitor
99.86
99.58
99.3
MLN-2238
NFE2L2
proteasome inhibitor
basic leucine zipper proteins, nuclear factor
(erythroid-derived 2)-like 2
99.91
99.82
Proteasome subunits, proteasome (prosome,
macropain) 26S subunit, non-ATPase, 1
PSMD1
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Proteasome subunits, proteasome (prosome,
macropain) subunit, beta type, 5
KD
KD
PSMB5
PSMA1
99.61
99.45
100
Proteasome subunits, proteasome (prosome,
macropain) subunit, alpha type, 1
PCL
PCL
HIF activator
N/A
N/A
Proteasome
inhibitor
99.72
Pc_selection is the connectivity score (from -100 to 100). A positive higher score
means more positive connection between the perturbagen and QCBT7.
2.7. QCBT7 and MG132 have similar transcriptional profiles
MG132, a peptide-aldehyde proteasome inhibitor, blocks the proteolytic activity of
the 26S proteasome by covalently binding to the 20S catalytic subunit [26]. Due to
its lack of specificity, it has only been used as a research tool to study the
ubiquitin-proteasome pathway. In a separate study, we performed Bru-seq of
MG132-treated HeLa cancer cells. Although a different cell line, we still identified
similarity in the CONCANNON_APOPTOSIS_BY_EPOXOMICIN_UP gene set
and WINTER_HYPOXIA_METAGENE gene set between QCBT7 and MG132.
QCBT7 and MG132 share 53 common upregulated enriched gene sets (FDR
< 0.001) from C2 curated gene sets (Figure 3A, Supplementary Table 2S).
Treatments with QCBT7 and MG132 induced 51 common genes contributing to
the enrichment of the CONCANNON_APOPTOSIS_BY_EPOXOMICIN_UP gene
set (Figure 3B-C). Moreover, their transcript expressions were strongly correlated
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(r = 0.69, p = 2.07e-08) (Figure 3D). STRING analysis indicates that the 51
common upregulated genes are involved in apoptotic signaling pathway, unfolded
protein response and ER stress. Furthermore, we observed 22 common genes in
the WINTER_HYPOXIA_METAGENE gene set from both QCBT7- and
MG132-treated samples, and their nascent transcript expressions also had a
strong correlation (r = 0.68, p = 5.64e-04) (Figure 3E-G). These 22 genes are
related to HIF-1 signaling pathway, glycolysis and cellular stress.
Figure 3. QCBT7 and MG132 have common enriched gene sets in proteasome
inhibition pathway and hypoxic response. (A) Venn diagram of all upregulated
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enriched gene sets between QCBT7 and MG132 (FDR < 0.001). (B) GSEA
enrichment plots of CONCANNON_APOPTOSIS_BY_EPOXOMICIN_UP gene
set in QCBT7 and MG132 Bru-seq samples. (C) 51 genes in common from the
EPOXOMICIN gene set. (D) Heatmap of the 51 common genes using log2FC (r =
0.69,
p
=
2.07e-08).
(E)
GSEA
enrichment
plots
of
WINTER_HYPOXIA_METAGENE gene set in two samples. (F) 22 genes in
common from HYPOXIA gene set. (G) Heatmap of the 22 common genes using
log2FC (r = 0.68, p = 5.64e-04).
Together, these results demonstrate that QCBT7 is similar to the proteasome
inhibitor MG132 at the transcription level and regulates redox signaling and ER
stress. GCLM, HMOX1, NQO1 and SQSTM1, from the 51 common genes in the
EPOXOMICIN gene set, are involved in redox signaling pathways. GCLM
encodes a glutamate-cysteine ligase modifier subunit, which is a rate-limiting
enzyme for glutathione synthesis. HMOX1 (heme oxygenase 1) responds to
cellular oxidative stress and catalyzes cleavage of heme in order to maintain
redox balance [27]. NQO1 (NAD(P)H quinone dehydrogenase 1) can also prevent
oxidative stress via reducing quinones to hydroquinones. In addition, SQSTM1
(sequestosome 1) mediates the activation of NF-κB signaling together with TNF
receptor-associated factor 6, and is a positive regulator of Nrf2 signaling [28].
CEBPB, DDIT3 and PPP1R15A are three ER stress-related genes induced by
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both QCBT7 and MG132. Proteasome inhibitors may stimulate CEBPB, a
transcription factor that interacts with DDIT3/CHOP, an ER stress biomarker, to
induce cellular stress and cause cancer cell death [27, 29]. PPP1R15A (GADD34)
sensitizes cells to proteasome inhibitors by promoting ER stress, reactive oxygen
species production and autophagy [30].
Proteasome inhibitors can regulate redox homeostasis and induce ER stress
[1, 27]. MG132 has been shown to activate the Nrf2-ARE signaling pathway that
protects cells from oxidative stress [31]. The inhibition of proteasome activity
caused by MG132 leads to the unfolded protein response and apoptosis [26, 32].
The findings in our study are consistent with previous reports on the mechanisms
of action of MG132. Moreover, they are similar to those of QCBT7 implied from
the STRING and GSEA results.
2.8. QCBT7 induces the accumulation of ubiquitylated proteins
To investigate whether QCBT7 inhibits proteasome activity as suggested from
Bru-seq analysis, immunoblot was performed using the antibody against the
ubiquityl group of ubiquitylated proteins in QCBT7-treated pancreatic cancer cells.
Ubiquitylated proteins are normally subjected to proteasome-mediated
degradation, but inhibition of the proteasome results in the accumulation of
slower-migrating ubiquitylated proteins. QCBT7 treatment resulted in the
dose-dependent accumulation of ubiquitylated proteins in both MIA PaCa-2 and
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PANC-1 cells (Figure 4). Similarly, both MG132 and ixazomib caused a significant
accumulation of ubiquitylated proteins (p < 0.005). QCBT7 showed a stronger
accumulating effect than MG132 in PANC-1, while ixazomib was the most potent
inducer of ubiquitylated proteins in both cell lines. These results support the
hypothesis that QCBT7 has proteasome inhibitory activity. Ixazomib, which
reversibly and selectively bind to the proteasome beta 5 subunit (PSMB5) of the
20S catalytic subunit, has been approved as an orally bioavailable proteasome
inhibitor for the treatment of multiple myeloma and is currently being tested in
combination with other standard-of-care treatments in clinical trials for solid
tumors [33, 34].
We also tested 2-chlorobenzoic acid (2-CBA), a potential by-product of
QCBT7 in the cellular reducing environment (Supplementary Scheme 1S). 2-CBA
did not cause accumulation of ubiquitylated proteins, suggesting that QCBT7 or
8TQ is the active proteasome inhibitory molecules.
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Figure 4. QCBT7 causes the accumulation of ubiquitylated proteins in MIA PaCa-2
and PANC-1 cell lines, similar to MG132 and ixazomib. Cells were treated with
QCBT7, MG132, ixazomib and 2-chlorobenzoic acid (2-CBA) for 24 h. MG132 and
ixazomib were used as positive controls. FC: fold change, relative expression of
the protein normalized to GAPDH expression. Data shown are representative of 3
independent experiments. Error bars represent standard deviation. * denotes p <
0.05, ** denotes p < 0.005 and *** denotes p < 0.0005 compared with DMSO
control.
2.9. QCBT7 increases the protein expression of genes related to glycolysis,
ER stress and hypoxia in pancreatic cancer cells
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To validate proteins and signaling pathways identified with the Bru-seq analysis,
we performed immunoblot of select genes related to glycolysis, ER stress and
hypoxia in MIA PaCa-2 and PANC-1 cells.
HK2 (hexokinase 2) and PFKFB4 (6-phosphofructo-2-kinase/fructose-2,
6-biphosphatase 4) are two glycolytic genes among the top 50 upregulated genes
(Figure 5A, Table 3). QCBT7 elevated HK2 and PFKFB4 protein expression
dose-dependently in both MIA PaCa-2 and PANC-1 cells (Figure 5B), and it
significantly increased their protein expression at 3.3 µM (Figure 5C, p < 0.05).
HK2 and PFKFB4 are related to hexose metabolic processes and their protein
expression increases under hypoxic conditions [35].
QCBT7 also induced CHOP expression, which is a protein biomarker for ER
stress (Figure 5B). Moreover, MG132 and ixazomib significantly increased
PFKFB4 and CHOP expression, but not HK2, suggesting that they also have
some differences in the downstream signaling due to potential distinct binding
sites of the proteasome.
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Figure 5. QCBT7 increases nascent RNA and protein expression of genes related
to glycolysis and ER stress in pancreatic cancer cells. (A) Trace diagrams of
nascent RNA expression of HK2 and PFKFB4. Blue represents QCBT7 treatment
and yellow represents the DMSO control. (B) Immunoblot of HK2, PFKFB4 and
CHOP in MIA PaCa-2 and PANC-1 cells after 24 h treatment. FC: fold change,
relative expression of the protein normalized to GAPDH expression. Data shown
are representative of 3 independent experiments. (C) Quantification of relative
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expression of each protein in 3 independent experiments. Data are reported as
mean ± standard deviation. * denotes p < 0.05 compared with DMSO control.
Furthermore, QCBT7 increased the expression of hypoxia-related proteins,
including HIF1A, SESN2, TRXR1, NQO1, HMOX1 and SLC7A11 (Figure 6). At
the nascent transcript level, NQO1, HMOX1 and SLC7A11 were dramatically
upregulated by QCBT7 (Figure 6A). HMOX1 and SLC7A11 protein expression
also increased significantly by 6.6
µM QCBT7, and NQO1 showed dramatic
increase in protein expression in PANC-1 but not MIA PaCa-2 cells (Figure 6B-C).
HIF1A, SESN2 and TRXR1 were upregulated by QCBT7 in both cell lines, and
HIF1A and SESN2 showed a dose-dependent increase (Figure 6B). MG132 and
ixazomib had similar effects on HIF1A, SESN2, HMOX1 and SLC7A11 protein
levels (Figure 6B-C). SESN2 (sestrin 2) is inducible by hypoxia and oxidative
stress conditions to balance metabolic homeostasis [36]. HMOX1 is a
downstream target of HIF1A, and is sensitive to cellular redox signaling [37]. In
addition, hypoxia affects glutathione metabolism [35]. TRXR1 (thioredoxin
reductase 1) was elevated by QCBT7 to possibly balance the increase of HIF1A
and maintain redox homeostasis. SLC7A11 (solute carrier family 7 member 11),
which regulates glutamate transport, also showed an increase expression with the
treatment [38].
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Figure. 6. QCBT7 increases nascent RNA and protein expression of genes related
to hypoxia in pancreatic cancer cells. (A) Trace diagrams of nascent RNA
expression of NQO1, HMOX1 and SLC7A11. Blue represents QCBT7 treatment
and yellow represents the DMSO control. (B) Immunoblot of HIF1A, SESN2,
TRXR1, NQO1, HMOX1 and SLC7A11 in MIA PaCa-2 and PANC-1 cells after 24 h
treatment. FC: fold change, relative expression of the protein normalized to
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GAPDH expression. Data shown are representative of 3 independent experiments.
(C) Quantification of relative expression of each protein in 3 independent
experiments. Data are reported as mean ± standard deviation. * denotes p < 0.05,
** denotes p < 0.005 and *** denotes p < 0.0005 compared with DMSO control.
Our immunoblot results of MG132 and ixazomib corroborate the previously
reported mechanisms of proteasome inhibitors in inducing hypoxia and ER stress.
MG132 increases the protein level of SESN2 due to the accumulation of
ubiquitylated SESN2, and it is dependent on Nrf-2 [36]. MG132, ixazomib and
bortezomib increase HMOX1 and CHOP expression level due to Nrf2 signaling
and ER stress [27, 31-34, 39]. Compared with MG132 and ixazomib, QCBT7 also
induces the expression of most proteins, such as PFKFB4, CHOP, HIF1A and
HMOX1, supporting their similar mechanisms (Figure 5-6). The signaling
pathways affected by QCBT7 have a strong correlation with its proteasome
inhibition activity.
Overall, HK2, PFKFB4, CHOP, HIF1A, SESN2, TRXR1, NQO1, HMOX1 and
SLC7A11 proteins are robustly upregulated in pancreatic cancer cells by QCBT7.
Most proteins show a dose-dependent increase in response to QCBT7, MG132
and ixazomib. These results indicate that QCBT7 induces hypoxia, ER stress and
glycolysis, and finally leads to cancer cell death, similar to MG132 and ixazomib.
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2.10. PFKFB4 is a potential biomarker to study proteasome inhibition
We identified that protein expression of PFKFB4 was significantly elevated by
MG132, ixazomib and QCBT7. Additionally, QCBT7 increased PFKFB4 protein
level back in the PFKFB4 knockdown MIA PaCa-2 cells (Supplementary Figure
6S). Our results show for the first time that PFKFB4 transcript and protein levels
can be used to evaluate the response to proteasome inhibitors in pancreatic
cancer. PFKFB4 is essential for cellular response to hypoxia, glucose metabolism
and cell survival, and its depletion in p53-deficient prostate cancer cells inhibits
tumor growth and cell survival [35, 40-42]. PFKFB4 is also recognized as an
autophagy regulator, relating to cancer cell death [43]. Most importantly, it may be
used as a new response regulatory protein to study the downstream signaling of
MG132, ixazomib and other proteasome inhibitors.
3. Conclusions
In this study, the STRING, GSEA and CMAP analyses of the novel compound
QCBT7 reveal that it induces transcriptional responses related to proteasome
inhibition, hypoxia, ER stress and glycolysis. Moreover, QCBT7 blocked the
degradation of ubiquitylated proteins, similar to MG132 and ixazomib. We
demonstrated that PFKFB4, HK2, CHOP, HIF1A, SESN2, TRXR1, NQO1,
HMOX1 and SLC7A11 protein levels increased upon QCBT7 treatment. MG132
and ixazomib also elevated PFKFB4, CHOP, HIF1A, SESN2, HMOX1 and
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