Sulfoxythiocarbamate S-4 inhibits HSP90 in human cutaneous squamous
cell carcinoma cells
Ying Zhang a
, Garrett C. VanHecke b
, Young-Hoon Ahn b
, Charlotte M. Proby a
Albena T. Dinkova-Kostova a,c,*
a Jacqui Wood Cancer Centre, School of Medicine, University of Dundee, Scotland, UK b Department of Chemistry, Wayne State University, Detroit, MI, USA c Department Pharmacology and Molecular Sciences and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
ARTICLE INFO
Keywords:
Cell cycle arrest
Electrophile
HSP90
Skin cancer
ABSTRACT
Cancer cells rely heavily on molecular chaperones, such as heat shock protein 90 (HSP90), and their co￾chaperones. The development of HSP90 inhibitors is an attractive therapeutic approach that has the potential
to affect multiple hallmarks of cancer. Such approach is particularly needed for tumors that carry large muta￾tional burdens, including cutaneous squamous cell carcinomas (cSCC). We previously identified sulfox￾ythiocarbamate S-4 as an HSP90 inhibitor. In this study, we investigated the mechanism(s) by which S-4
compromises the viability of human cSCC cells. S-4 inhibits HSP90 and causes depletion of its clients HER2, a
tyrosine kinase oncoprotein, and Bcl-2, an anti-apoptotic protein. The decrease in Bcl-2 is accompanied by cy￾tochrome c release from mitochondria into the cytoplasm, suggesting apoptosis. In the surviving cells, depletion
of the HSP90 clients cyclin D and CDK4 by S-4 prevents phosphorylation of the retinoblastoma protein Rb and
the release of transcription factor E2F, inhibiting G1-S cell cycle progression and cell division. These findings
illustrate the comprehensive effectiveness of S-4 and encourage future development of compounds of this type for
cancer prevention and treatment.
1. Introduction
Heat shock protein 90 (HSP90) facilitates the correct folding and
stability of a broad range of ~200 “client” proteins. (Taipale et al.,
2010). In cancer cells, HSP90 acts as a molecular chaperone for
numerous oncogenic proteins and is essential for their activity and sta￾bility, particularly for those that are activated by mutation(s) or over￾expression (Taldone et al., 2020; Whitesell and Lindquist, 2005). Thus,
the development of inhibitors of HSP90 is an attractive strategy for
cancer treatment (Trepel et al., 2010). Moreover, inhibition of HSP90
shows promise for overcoming drug resistance that frequently limits
successful cancer treatment, and for improving the efficacy of immu￾notherapy (Kryeziu et al., 2019).
Cutaneous squamous cell carcinomas (cSCC), which are most
commonly caused by recreational or occupational exposure to solar ul￾traviolet radiation (UVR), represent some of the most frequently diag￾nosed keratinocyte skin cancers, and the most highly mutated human
malignancies. Although surgical excision, radiotherapy, and
chemotherapy are effective against primary tumors, mortality for met￾astatic cSCC is as high as 70% (Burton et al., 2016). Comprehensive
characterization studies of the genomic changes that drive development
of cSCC in both humans and mice have revealed mutations in numerous
oncogenes and tumor suppressor genes (Chitsazzadeh et al., 2016;
Knatko et al., 2017; South et al., 2014), implying that therapies that
target individual oncoproteins are unlikely to succeed, and global ap￾proaches are needed for the prevention and treatment of these cancers.
Topical treatment of the skin of mice with the HSP90 inhibitor
17-N-allylamino-17-demethoxygeldanamycin (17-AAG) prevents the
development of UVR-induced SCC, and is accompanied by a decrease in
UVR-induced hyperplasia of the skin, and lower levels of a number of
HSP90 client proteins without any local or systemic toxicity (Singh
et al., 2015). These preclinical observations suggest that HSP90 in￾hibitors could be developed as topical agents for prevention of cSCC.
Using click chemistry, we previously found that the mildly electro￾philic compound sulfoxythiocarbamate alkyne (STCA) binds to cysteine
residues of Kelch-like ECH associated protein 1 (Keap1) (Ahn et al.,
* Corresponding author. Division of Cellular Medicine, School of Medicine, Jacqui Wood Cancer Centre, James Arnott Drive, Dundee, DD1 9SY, Scotland, United
Kingdom.
E-mail address: [email protected] (A.T. Dinkova-Kostova).
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

https://doi.org/10.1016/j.ejphar.2020.173609

Received 2 June 2020; Received in revised form 18 September 2020; Accepted 28 September 2020
European Journal of Pharmacology 889 (2020) 173609
Fig. 1. S-4 induces HSP70. (A) Chemical structure of S-4. (B,C) Viability of SCC IC1 cells (B) and normal human keratinocyte (NHK) cells (C) that had been left
untreated, or had been treated with vehicle (0.1% acetonitrile) or S-4 (2.5-, 5- or 7.5 μM) for the indicated periods of time. *, p < 0.05 relative to vehicle-treated cells
(Student’s t-test). (D) Immunoblotting analysis for HSP70 in SCC IC1 cells that had been exposed to increasing concentrations of S-4 for 24 h. GAPDH served as the
loading control. (E) Immunoblotting analysis for HSP70 and phosphorylated HSF1 (at S326) in cells that had been exposed to S-4 (7.5 μM) for increasing periods of
time. β-actin served as the loading control. Results are representative of 2 independent experiments.
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
2010) to activate transcription factor nuclear factor erythroid 2
p45-related factor 2 (Nrf2), which in turn regulates the expression of a
network of genes encoding cytoprotective proteins (Cuadrado et al.,
2019). Genetic or pharmacologic activation of Nrf2 is protective against
UVR-induced skin damage in mice and humans (Dinkova-Kostova et al.,
2008; Knatko et al., 2015; Talalay et al., 2007), and reduces the devel￾opment of cSCC caused by chronic exposure to solar-simulated UVR in
mice (Knatko et al., 2015, 2016), suggesting that pharmacologic Nrf2
activators are good candidates for photoprotective agents. A mass
spectrometry-based proteomic approach showed that in addition to
Keap1, STCA also binds to HSP90 in cells (Ahn et al., 2010; Zhang et al.,
2011) and covalently modifies cysteine residues of purified recombinant
HSP90 in vitro, forming stable thiocarbamate adducts (Zhang et al.,
2014). A cell-based screen of 19 sulfoxythiocarbamate derivatives
identified compound S-4 (Fig. 1A) as an HSP90 inhibitor, which de￾stabilizes multiple HSP90 client proteins and decreases proliferation of
breast cancer cells (Zhang et al., 2014). Among the HSP90 clients
downregulated by S-4 in these cells was HER2 (gene name ERBB2).
Curiously, molecular similarities, including mutations in HER2, have
been observed between cSCC and specific subtypes of breast cancer,
suggesting common molecular vulnerabilities (Chitsazzadeh et al.,
2016). In the present study we address the mechanism(s) by which S-4
compromises cell viability using early passages of patient-derived cSCC
cells as a model system.
2. Materials and methods
2.1. Materials
AUY922 (NVP-AUY922) was from ApexBio Technology. S-4 was
synthesized as described (Zhang et al., 2014). The identity and purity
(>94%) of S-4 were determined by 1
H- and 13C-NMR spectroscopy and
liquid chromatography-mass spectrometry (LC-MS). Spectra are shown
in Supplemental Fig. 1A. For cell culture experiments, AUY922 was
dissolved in DMSO and S-4 was dissolved in acetonitrile. Both solutions
were then diluted (1:1000) in the cell culture medium. The concentra￾tion of solvent in the cell culture medium was maintained at 0.1%. All
other general chemicals and reagents were of analytical grade and ob￾tained from Sigma-Aldrich.
2.2. HSP90β expression and purification
A plasmid encoding human GST-HSP90β fusion protein (Zhang et al.,
2014) was transformed into E. coli BL21 (DE3) codon plus cells. Cells
were grown at 37 ◦C in LB media until OD600 of 0.6–0.8, then induced at
37 ◦C with isopropyl β-d-1-thiogalactopyranoside (IPTG, 1 mM) for 4 h.
Cell pellets were collected by centrifugation and sonicated in PBS. Cell
lysates were clarified by centrifugation at 17,700 x g. The supernatant
was loaded onto Glutathione Sepharose 4 FF (GE healthcare) for affinity
purification. Bound protein was cleaved by Precision Protease at 4 ◦C
overnight, and HSP90β was collected in the flow-through fraction.
Protein purity was confirmed following SDS-PAGE and Coomassie blue
staining.
2.3. Cysteine modification of HSP90β by S-4
Purified HSP90β was dialyzed against 40 mM HEPES, pH 7.5, 20 mM
KCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Dialysed Hsp90β
(1 μM) was incubated with S-4 (15 μM) or DMSO (vehicle for S-4) in 50
μl of buffer (40 mM HEPES, pH 7.5, 20 mM KCl, 1 mM TCEP), at room
temperature, for 2 h. The samples were then mixed with NuPAGE™ LDS
Sample Buffer (Thermo Scientific), incubated at room temperature for
30 min, and gel-purified using NuPAGE™ 4–12% Bis-Tris gel and
NuPAGE™ MOPS SDS Running Buffer (Thermo Scientific). The gel was
stained with InstantBlue™ (Expedeon) and the Hsp90β band was
excised, reduced with TCEP, alkylated with 2-iodoacetamide (IAA), in￾gel digested with trypsin, and subjected to analysis by liquid
chromatography-mass spectrometry (LC-MS) on an Orbitrap Velos
equipped with Easy spray source and Ultimate 3000 UPLC.
2.4. Cell culture
The establishment of the human cutaneous squamous cell carcinoma
cell lines SCC IC1 and SCC IC12 has been previously described (Proby
et al., 2000). Cell lines were cultured in RM+ media with the following
composition: a mixture of DMEM:Ham’s F12 (3:1) (Thermo Scientific)
media supplemented with 10% fetal bovine serum (FBS, Thermo Sci￾entific), 0.4 μg/ml hydrocortisone (Sigma), 5 μg/ml insulin (Sigma), 10
ng/ml epidermal growth factor (EGF, Serotec), 5 μg/ml transferrin
(Sigma), 8.4 ng/ml cholera toxin (Sigma) and 13 ng/ml liothyronine
(Sigma). Normal Human Keratinocyte (NHK) cells were isolated from
breast or abdominal skin of human subjects after informed consent,
initially grown in the presence of a mitotically inactivated NIH3T3
fibroblast feeder layer as described (Rheinwald, 1980), and cry￾opreserved. Frozen NHK cells were rapidly thawed in a 37 ◦C water bath
and cultured overnight in RM- (same composition as RM+, but without
EGF) media. On the next day, the media was replaced with RM+ media.
Cells were maintained in a humidified atmosphere at 37 ◦C and 5% CO2.
2.5. ATP-binding assay
SCC IC1 cells (0.5 × 106
) growing in 6-cm dishes were treated for 24
h with either 0.1% (v/v) acetonitrile (vehicle for S-4), 7.5 μM S-4, 0.1%
DMSO (vehicle for AUY922) or 0.1 μM AUY922. Using a scraper, cells
were collected into 300 μl of lysis buffer (10 mM Tris pH 7.5, 150 mM
NaCl, 0.25% NP40, with one protease inhibitor tablet (Roche) per 10.0
ml of buffer), and the cell suspension was frozen, thawed, and lysed for
30 min at 4 ◦C. ATP-agarose beads (Jena Bioscience, AC-101S) were
washed with incubation buffer (10 mM Tris pH 7.5, 150 mM NaCl, 20
mM MgCl2, 0.05% NP40, 1 mM DTT). The beads suspension (30 μl) was
mixed with 200 μg of protein in a final volume of 1.25 ml of incubation
buffer, and incubated rotating overnight at 4 ◦C. The beads were then
collected by centrifugation and washed three times with incubation
buffer. Following addition of 10 μl of sodium dodecyl sulfate (SDS)
loading buffer and 40 μl of incubation buffer, the samples were heated at
100 ◦C for 5 min. The beads were pelleted by centrifugation, and the
supernatants were collected and subjected to immunoblotting analysis.
2.6. Immunoblotting
Cells growing on 6-well plates were lysed in 150 μl of non-reducing
sample buffer (50 mM Tris-HCl pH 6.8, 2% (w/v) sodium dodecyl sulfate
(SDS), 10% (v/v) glycerol, and 0.02% (w/v) Bromophenol blue). Whole￾cell lysates were then collected in Eppendorf tubes, boiled at 100 ◦C for
5 min, and sonicated using Vibra-Cell ultrasonic processor (Sonic) for
20 s at 20% amplitude. The BCA assay (Thermo) was used to determine
protein concentrations. 2-Mercaptoethanol (Sigma) was added to a final
concentration of 6% (v/v). Proteins were resolved by SDS/PAGE, and
then transferred to 0.45 μm nitrocellulose (NC) membranes (Thermo
Scientific). Solutions of primary antibodies were prepared in 3% BSA
(antibodies against phospho-proteins) or 5% milk (all other) in PBST.
The following antibodies and dilutions were used: mouse monoclonal
anti-HSP70, 1:1000, StressMarq, SMC-100B; rabbit monoclonal anti￾phospho-HSF1 (Ser326), 1:5000, Abcam, ab76076; rabbit monoclonal
anti-Hsp90β, 1:5000, Abcam, ab203085; rabbit polyclonal anti-HER2,
1:500, Millipore, 06-562; rabbit monoclonal anti-Bcl-2, 1:1000, CST,
2870; rabbit monoclonal anti-cytochrome c, 1:1000, CST, 11940T;
rabbit monoclonal anti-CDK4, 1:1000, CST, 12790T; rabbit monoclonal
anti-cyclin D1, 1:1000, CST, 2978T; rabbit polyclonal anti-phospho-Rb
(Ser795), 1:1000, CST, 9301T; rabbit monoclonal anti-phospho-Rb
(Ser608), 1:1000, CST, 8147T; rabbit polyclonal anti-GAPDH, 1:5,000,
Sigma, G9545; rabbit polyclonal anti-VDAC-1, 1:10000, Abcam,
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
4
ab154856; mouse monoclonal anti-β-actin, 1:20000, Sigma, A5441.
Blocked NC membranes were incubated with the primary antibody so￾lutions in 50-ml tubes at 4 ◦C overnight, with continuous rotation. After
this, the NC membranes were washed and incubated with the corre￾sponding secondary antibodies (horseradish peroxidase (HRP)-conju￾gated goat anti-rabbit or goat anti-mouse antibodies, 1:5000, BioRad).
Thermo Scientific SuperSignal West Dura Extended Duration Substrate
was used for signal development. For the experiment shown in Fig. 2,
IRDye fluorescent dyes conjugated to secondary antibodies (GAM/GAR-
800CW, 1:15000, LI-COR) were used. Image capture and analysis were
done using the Odyssey® CLx image system and Image Studio software
(LI-COR).
2.7. Cell viability assay
Cells (5 × 103 per well) were seeded in white flat-bottomed 96-well
plates (Nunc). On the next day, cells were treated with 0.1% (v/v)
acetonitrile (vehicle control) or increasing concentrations of S-4 for 24,
48 or 72 h. Four h before the end of each treatment, 10 μl of alamarBlue
dye solution (AbdSerotec) was added to the cell culture media (100 μl)
within each well, and the plates were returned to the incubator. After 4
h, the fluorescence of the reduced probe was determined (ex. 560 nm/
em. 590 nm, SpectraMax M2, Molecular Devices), from which the cell
viability was calculated. For both cell types, the experiment was done on
two separate occasions, each time using 8 replicate wells of cells.
2.8. Cell proliferation assay
Cell proliferation was followed for 3 days using the CellTrace™ CFSE
(carboxyfluorescein diacetate succinimidyl ester, Thermo Scientific).
Briefly, cells were labeled with 2.5 μM CFSE according to the manu￾facturer’s protocol, and seeded at a density of 2.5 × 105 per well in 6-
well plates. On the next day, cells were treated with 0.1% (v/v) aceto￾nitrile (vehicle control) or increasing concentrations of S-4. At the
indicated time points, the cells were collected and analyzed using BD
LSRFortessa™ cell analyzer with 488 nm excitation and emission filters
appropriate for fluorescein to measure CFSE fluorescence. This experi￾ment was done twice.
2.9. Cell cycle analysis
Cells were seeded at a density of 2.5 × 105 per well in 6-well plates.
On the next day, cells were treated with 0.1% (v/v) acetonitrile or
increasing concentration of S-4 for the indicated time periods. After
treatment, the cells were collected and fixed with 70% ethanol at -20 ◦C
overnight. Cells were then washed with PBS and stained with PI (40 μg/
ml) in the presence of RNase A (100 μg/ml) at 37 ◦C for 30 min in the
dark. The DNA content was detected using BD LSRFortessa™ cell
analyzer. The data were analyzed in FlowJo. The experiment was done
on three separate occasions.
2.10. Data analysis
All quantitative data are represented graphically as mean values ± 1
standard deviation (S.D.). The differences between groups were deter￾mined by Student’s t-test.
3. Results
3.1. S-4 upregulates HSP70 in cSCC cells
The cell line SCC IC1 was originally isolated from the primary site of
a moderately differentiated metastatic squamous cell carcinoma that
had been formed on the right temple of an immunocompetent 77-year￾old male patient. SCC IC1 cells have well characterized mutational
spectrum comprised of multiple UV radiation signature mutations,
including mutations in TP53 (H179Y, R248W), NOTCH1 (N1809H),
NOTCH2 (P1913S) and ERBB2 (E1068K) (South et al., 2014). For some
experiments, we also used SCC IC12 cells isolated from a moder￾ately/poorly differentiated squamous cell carcinoma that had been
formed on the left calf of an immunocompetent 87-year-old female
patient.
Monitoring the activation of transcription factor heat shock factor 1
(HSF1), the master regulator of the heat shock response, by the
enhanced expression of its downstream target proteins such as heat
shock protein 70 (HSP70), represents a mechanistically unbiased
approach to screen individual compounds and chemical libraries for
identification of HSP90 inhibitors (Santagata et al., 2012). Induction of
HSP70 correlates with degradation of HSP90 client protein in peripheral
blood lymphocytes and xenograft tumors (Mehta et al., 2011), and is
used as a pharmacodynamic marker in clinical trials with HSP90 in￾hibitors (Shimomura et al., 2019). Therefore, to begin evaluating the
ability of S-4 to inhibit HSP90 in SCC IC1 cells, we used induction of
HSP70 as the first endpoint. The concentration range of S-4 was chosen
based on a cell viability assay, which showed 20- and 50% loss of
viability of SCC IC1 cells at 72 h post-exposure to 5- and 7.5 μM S-4,
respectively (Fig. 1B). Importantly, these concentrations of S-4 were less
toxic to cultured normal human keratinocytes (NHK) cells, and >90- and
80% of them were viable 72 h after 5- and 7.5 μM S-4 treatment,
respectively (Fig. 1C). We found that the levels of HSP70 increased in a
concentration-dependent manner 24 h after exposure of SCC IC1 cells to
S-4 (Fig. 1D). Similar results were obtained in SCC IC12 cells (Supple￾mental Fig. 2A). A time-course experiment following exposure of SCC
IC1 cells to 7.5 μM S-4 further showed that induction of HSP70 was
time-dependent, and among the time points tested, was maximal at the
24-h time point (Fig. 1E). The induction of HSP70 was preceded by a
transient increase in the levels of phosphorylated HSF1 at S326, a
hallmark of HSF1 activation (Fig. 1E). These results are in close agree￾ment with the ability of S-4, and other structurally related sulfox￾ythiocarbamates, to induce HSP70 in breast cancer cell lines (Zhang
et al., 2014).
3.2. S-4 binds to cysteine residues of HSP90β
Although induction of HSP70 can be a consequence of HSP90 inhi￾bition by compounds that bind to the N-terminus of the chaperone, it can
also occur independently of HSP90. Wang et al. (2017) have reported
that the cellular phenotypes resulting from treatments with N-terminal
Fig. 2. S-4 does not interfere with the ability of HSP90β to bind ATP. SCC
IC1 cells were exposed for 24 h to acetonitrile (0.1%), S-4 (7.5 μM in 0.1%
acetonitrile), DMSO (0.1%), or AUY922 (0.1 μM in 0.1% DMSO), lysed and
subjected to ATP pull-down using ATP-agarose beads. The levels of HSP90β,
HSP70 and β-actin were determined by immunoblotting. Results are represen￾tative of 2 independent experiments.
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
5
HSP90 inhibitors (e.g. AUY922 or 17-AAG) differ from the phenotype
caused by a knockdown of HSP90, whereas those consequent to treat￾ments with C-terminal HSP90 inhibitors, phenocopy HSP90-deficient
cells. We have previously shown that STCA forms covalent thio￾carbamate adducts with cysteine residues in the middle domain of
HSP90 (Zhang et al., 2014). To test the possibility that S-4 acts by a
similar mechanism, we produced recombinant purified human HSP90β
and incubated it with S-4 or its vehicle control for 2 h. Following gel
purification and in-gel tryptic digestion of the modified protein, the
resulting peptides were subjected to mass spectrometry analysis. Five
peptides were identified in the S-4-treated sample, which were absent in
the vehicle-treated sample (Table S1), collectively revealing that S-4
formed covalent adducts with C521, and C589/C590 of HSP90β.
Notably, C521, C589, and C590 are located in the middle domain of
HSP90, suggesting that their modification may interfere with the ability
of HSP90 to bind its co-chaperones and/or client proteins, but not with
its ATP-binding ability. It was still theoretically possible however that S-
4 binds non-covalently to the N-terminal domain of HSP90. To test for
this possibility, we performed an ATP-agarose pull-down of lysates from
SCC IC1 cells that had been treated with S-4 or its vehicle control. As
expected, treatment with the N-terminal inhibitor AUY922, which was
used as positive control, abolished binding of HSP90 to ATP (Fig. 2). By
contrast, ATP binding of HSP90 was not altered by S-4 (Fig. 2). Because
ATP binds to the N-terminal domain of HSP90, this result further con￾firms that the mode of action of S-4 is distinct from that of N-terminal
inhibitors.
3.3. S-4 destabilizes the HSP90 client proteins HER2 and Bcl-2
Next, we tested the functional consequences of S-4 binding to HSP90.
SCC IC1 cells carry mutant HER2, a tyrosine kinase oncoprotein, which
has been shown to interact strongly with HSP90, emphasizing the
importance of the chaperone for HER2 stability (Taipale et al., 2012).
Consistent with HSP90 inhibition, exposure of SCC IC1 cells to S-4 for
24 h led to a concentration-dependent decrease in the levels of immu￾nologically detectable HER2 (Fig. 3A). A time-course experiment further
revealed a clear decrease of HER2 at the 8- and the 24-h time-points
(Fig. 3B). A concentration-dependent decrease in the levels of HER2
after exposure to S-4 was similarly observed in SCC IC12 cells (Supple￾mental Fig. 2B). Pharmacological inhibition of HSP90 has been shown to
cause downregulation of another HSP90 client, Bcl-2 (Gallerne et al.,
2013), a protein of critical importance for the integrity of the mito￾chondrial membrane (Harris and Thompson, 2000). In agreement with
the dependence of Bcl-2 on HSP90 and HSP90 inhibition by S-4, we
found that the levels of Bcl-2 were reduced in cells treated with 7.5 μM
S-4, especially at the early (2- and 4-h) time points (Fig. 3C).
3.4. S-4 promotes cytochrome c release
Bcl-2 is an anti-apoptotic protein, which together with Bcl-xL, in￾hibits Bax-mediated apoptosis, promoting cell survival (Zha et al.,
1996). To further explore the functional consequences of S-4 treatment,
we considered the possibility that, by destabilizing Bcl-2, S-4 may
release its inhibitory effect on Bax. This would allow Bax to form pores
on the mitochondrial membrane, in turn leading to cytochome c release
and activation of apoptosis. A sub-cellular fractionation experiment
showed that exposure to S-4 led to a concentration-dependent cyto￾chrome c release in the cytoplasm (Fig. 3D), suggesting induction of
apoptosis via the intrinsic apoptotic pathway.
3.5. S-4 causes cell cycle arrest
Treatment with S-4 for 24 h caused inhibition in cell proliferation in
a concentration-dependent manner (Fig. 4A), and this effect was even
more pronounced at 48 h post-treatment in both SCC IC1 (Fig. 4B) and
SCC IC12 (Supplemental Fig. 3A) cells. One possible explanation was
that those cells that survived apoptosis were undergoing cell cycle ar￾rest. To address this possibility, cell cycle analysis was performed. This
analysis showed that S-4 caused a concentration-dependent arrest at the
G0/1 phase of the cell cycle at 24 h following exposure to S-4 (Fig. 4C).
Overall, from three independent experiments, at 24 h, compared to
vehicle-treatment, the cell population in G0/1 was increased by 2.1-fold
(P = 0.05) and 2.8-fold (P = 0.036) after treatment with 5- and 7.5 μM S-
4, respectively (Fig. 4D). Similar results were obtained in SCC IC12 cells
(Supplemental Fig. 3B).
During early G1, the transcription factor E2F is inhibited by inter￾action with retinoblastoma susceptibility protein Rb (Fig. 5A). Activa￾tion of the cyclin D-CDK4/6 protein complex results in the
Fig. 3. S-4 destabilizes the HSP90 client proteins HER2 and Bcl-2. (A,B)
Immunoblotting analysis for HER2 in cells that had been exposed to increasing
concentrations of S-4 for 24 h (A) or to 7.5 μM of S-4 for the indicated periods of
time (B). (C) Immunoblotting analysis for Bcl-2 in cells that had been exposed
to S-4 (7.5 μM) for the indicated periods of time. The band in the red square
corresponds to Bcl-2; the faster-migrating band is a non-specific protein. (D)
Immunoblotting analysis for cytochrome c in the cytoplasmic fraction of cells
that had been exposed to increasing concentrations of S-4 for 8 h. β-actin served
as the loading control. Sub-cellular fractionation was done using standard
protocol from Abcam. The absence of detectable VDAC-1 served as evidence for
the absence of mitochondrial contamination in the purified cytoplasmic frac￾tion. In all experiments, acetonitrile (0.1%) served as the vehicle control and
β-actin served as the loading control. Immunoblots are representative of 2 in￾dependent experiments. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
phosphorylation of Rb at multiple sites, which then releases E2F, in turn
activating the transcription of genes that allow G1-S phase progression
(Knudsen and Knudsen, 2008). Importantly, E2F was identified as a key
transcriptional driver in the early stages of the development of cSCC in
both humans and mice (Chitsazzadeh et al., 2016). Both CDK4 and
cyclin D1 are HSP90 client proteins (Vaughan et al., 2006). Consistent
with HSP90 inhibition, the levels of CDK4 and cyclin D1 decreased in a
time-dependent manner in S-4-treated cells (Fig. 5B). In agreement with
the decreased levels of CDK4 and cyclin D1, the levels of phosphorylated
Rb at S608 and S795 were also lower in S-4-treated cells (Fig. 5B).
Fig. 4. S-4 causes cell cycle arrest. (A, B) Flow cytometry analysis of cells that had been labeled with CFSE and treated with vehicle (0.1% acetonitrile) or
increasing concentrations of S-4 for either 24- (A) or 48- (B) h. (C) Cell cycle analysis of cells that had been left untreated, or treated with vehicle (0.1% acetonitrile)
or S-4 (5- or 7.5 μM) for the indicated periods of time. Results are representative of 3 independent experiments. (D) Combined results of cell cycle analysis following
treatment with vehicle (0.1% acetonitrile) or S-4 (5- or 7.5 μM) for 24 h. P = 0.05 for cells treated with 5 μM S-4 compared to vehicle-treated cells; P = 0.036 for cells
treated with 7.5 μM S-4 compared to vehicle-treated cells (Student’s t-test).
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
Together, these experiments suggest that S-4 causes destabilization of
cyclin D1 and CDK4, consequently decreasing the phosphorylation of Rb
and promoting G1-S cell cycle arrest.
4. Discussion
A large number of HSP90 inhibitors have been developed, and
several have been tested in clinical trials (Jhaveri et al., 2012). Most
have demonstrated limited clinical efficacy and unacceptable adverse
events, such as ocular toxicities that are likely caused by photoreceptor
cell death consequent to sustained HSP90 inhibition in the retina (Aguila
et al., 2014; Zhou et al., 2013). A recent clinical trial in patients with
advanced solid tumors with the orally-administered pyrazolo[3,4- b]
pyridine derivative TAS-116 has identified specific dosing regimens
Fig. 5. S-4 causes depletion of the HSP90
client proteins CDK4 and cyclin D1, and
inhibition of phosphorylation of the Rb
protein. (A) The cyclin D-CDK4/6 protein
complex phosphorylates Rb, which releases
E2F to activate the transcription of genes
that allow G1-S phase progression. (B)
Immunoblotting analysis for CDK4, cyclin
D1, and Rb protein phosphorylated at S608
and S795 in cells that had been exposed to S-
4 (7.5 μM) for the indicated periods of time.
β-actin served as the loading control. In B,
CDK4 was detected on the lower section of
the same membrane shown in Fig. 1E,
whereas HSP70 was detected using the
membrane upper section; Cyclin D1 was
detected on the lower section of the same
membrane shown in Fig. 1E, whereas pHSF1
(S326) was detected using the membrane
upper section; pRb(S795) was detected on
the lower section of the same membrane
shown in Fig. 3B, whereas HER2 was
detected on the membrane upper section,
and thus each of these two blots share the
same β-actin loading control. Immunoblots
are representative of 2 independent
experiments.
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
with promising safety and efficacy profiles, forming the basis for the
current testing of this HSP90 inhibitor in patients with non-small cell
lung cancer, gastrointestinal stromal tumors, and HER2-positive breast
cancer (Shimomura et al., 2019).
Following from our previous findings that sulfoxythiocarbamate
derivatives induce transcription factor Nrf2 (Ahn et al., 2010) and
inhibit HSP90 and the proliferation of breast cancer cells (Zhang et al.,
2014), in this study, we investigated the mechanism(s) by which the
sulfoxythiocarbamate S-4 compromises the viability of cancer cells using
two patient-derived primary cSCC cell lines. Based on the current re￾sults, we propose a model, which is summarized in Fig. 6. S-4 inhibits
HSP90, causing depletion of its client proteins, including Bcl-2, cyclin
D1 and CDK4. Depletion of Bcl-2 allows Bax to form pores on the
mitochondrial membrane, triggering cytochrome c release. Depletion of
the HSP90 clients cyclin D1 and CDK4 by S-4 does not allow phos￾phorylation of Rb and the release of E2F and prevents G1-S cell cycle
progression. Together, these processes lead to compromised cell
viability.
This model is based on our new findings that treatment with S-4
decreases the levels of Bcl-2, cyclin D1 and CDK4, all of which are well￾established HSP90 clients, and the known functions of these proteins in
apoptosis and the cell cycle. It should be noted however that S-4 is not a
selective inhibitor of HSP90, and other potential mechanisms could also
be contributing to the observed effects. By virtue of its sulfhydryl
reactivity, S-4 may also bind to other cellular proteins, similarly to the
closely related sulfoxythiocarbamate alkyne (STCA) (Ahn et al., 2010).
Indeed, S-4 activates transcription factor Nrf2, as evidenced by the in￾crease in the levels of the classical Nrf2-target protein, NAD(P)H:
quinone oxidoreductase 1 (NQO1) (Zhang et al., 2014). Importantly,
Nrf2 activation by compounds of this type requires lower concentrations
than the concentrations that inhibit HSP90/induce HSP70 (Zhang et al.,
2011) as Keap1 is endowed with highly reactive cysteine sensors (Din￾kova-Kostova et al., 2002). Pharmacologic Nrf2 activation results in
comprehensive multi-organ cytoprotection, including protection of the
retina (Gao and Talalay, 2004; Pitha-Rowe et al., 2009; Tanito et al.,
2005), and therefore could counteract some of the negative conse￾quences of HSP90 inhibition. Thus, the ability of S-4 to both activate
Nrf2 and inhibit HSP90 offers the advantage of protecting normal cells
and tissues, whilst causing cell cycle arrest of cancer cells, which are
much more reliant on HSP90 than normal cells for growth and survival.
CRediT authorship contribution statement
Ying Zhang: Investigation, Data Analysis, Writing – original draft,
Writing – review & editing, All authors read and approved the final
manuscript. Garrett C. VanHecke: Reagents. Young-Hoon Ahn:
Conceptualization, Writing – review & editing. Charlotte M. Proby:
Writing – review & editing, Reagents.
Declaration of competing interest
A.T.D-K. is a member of the Scientific Advisory Board of Evgen
Pharma, and a consultant for Aclipse Therapeutics and Vividion
Therapeutics.
Acknowledgments
We thank Wenzhang Chen and the FingerPrints Proteomics Facility
of the University of Dundee School of Life Sciences for mass spectrom￾etry analysis. We acknowledge with gratitude the financial support of
the Biotechnology and Biological Sciences Research Council (BBSRC,
Project Grant BB/J007498/1) and Cancer Research UK (C20953/
A18644).
Fig. 6. Proposed model for the mechanism by which S-4 decreases the viability of SCC IC1 cells. S-4 inhibits HSP90, causing depletion of its client proteins,
including Bcl-2, cyclin D1 and CDK4. The decrease in Bcl-2 allows Bax to form pores on the mitochondrial membrane, triggering cytochrome c release, suggesting
apoptosis. Depletion of the HSP90 clients cyclin D1 and CDK4 prevents phosphorylation of Rb and the release of E2F, inhibiting G1-S cell cycle progression.
Y. Zhang et al.
European Journal of Pharmacology 889 (2020) 173609
9
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2020.173609.
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