Cancer Letters 343 (2014) 286294

Contents lists available at ScienceDirect

Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Targeting Mcl-1 for multiple myeloma (MM) therapy: Drug-induced

generation of Mcl-1 fragment Mcl-1128350 triggers MM cell death
via c-Jun upregulation
Fengjuan Fan a, Giovanni Tonon b, Muhammad Hasan Bashari a, Sonia Vallet a, Elena Antonini b,
Hartmut Goldschmidt d, Henning Schulze-Bergkamen a, Joseph T. Opferman e, Martin Sattler c,
c
Kenneth C. Anderson , Dirk Jger a, Klaus Podar a,
a

Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany
Functional Genomics of Cancer Unit, Division of Molecular Oncology, San Raffaele Scientic Institute, Milan, Italy
c
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
d
Section Multiple Myeloma, Department of Internal Medicine V, National Center for Tumor Diseases (NCT), University of Heidelberg, Heidelberg, Germany
e
St. Jude Childrens Research Hospital, Memphis, TN, USA
b

a r t i c l e

i n f o

Article history:
Received 27 July 2013
Accepted 27 September 2013

Keywords:
Mcl-1
c-Jun
Multiple myeloma
Apoptosis
MEFs
Glioblastoma

a b s t r a c t
Myeloid cell leukemia-1 (Mcl-1, HGNC: 6943), a pro-survival member of the Bcl-2 family, plays a
crucial role in Multiple Myeloma (MM) pathogenesis and drug resistance, thus representing a promising therapeutic target in MM. A novel strategy to inhibit Mcl-1 activity is the induction of
ubiquitin-independent Mcl-1 degradation. Our own and other previous studies have demonstrated
caspase-dependent generation of a 28 kDa Mcl-1 fragment, Mcl-1128350, which inhibits MM cell
proliferation and survival. Here, we show that similar to bortezomib, the novel proteasome inhibitors carlzomib and ixazomib, as well as staurosporine and adaphostin, induce the generation of
Mcl-1128350 in MM cells. Next, the molecular sequelae downstream of Mcl-1128350, which mediate
its pro-apoptotic activity, were delineated. Surprisingly, we observed nuclear accumulation of druginduced or exogenously overexpressed Mcl-1128350, followed by elevated mRNA and protein levels
of c-Jun, as well as enhanced AP-1 reporter activity. Moreover, drug-induced AP-1 activity was
blocked after introducing a point mutation into the highly conserved Mcl-1 caspase-cleavage site
Asp127, but not Asp157. Consequently, drug-triggered cell death was signicantly decreased in
MM cells transfected with Mcl-1 D127A, but not with Mcl-1 D157A. Consistent with these data,
treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt, but
not Mcl-1D/null murine embryonic broblasts (MEFs). Transfection of a plasmid carrying Mcl-1wt
into Mcl-1D/null MEFs restored bortezomib-induced Mcl-1 fragmentation, c-Jun upregulation and
AP-1 reporter activity. Finally, our data indicate that drug-induced generation of a pro-apoptotic
Mcl-1 fragment followed by c-Jun upregulation may also be a novel therapeutic approach in other
tumor entities.
2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Multiple myeloma (MM), the second most common blood cancer in adults, is characterized by bone marrow (BM) plasmacytosis, monoclonal protein in blood and/ or urine, bone lesions, renal
Corresponding author. Address: Medical Oncology, National Center for Tumor
Diseases (NCT), University of Heidelberg and German Cancer Research Center
(DKFZ), Im Neuenheimer Feld #460, 69120 Heidelberg, Germany. Tel.: +49 (0) 6221
56 38689; fax: +49 (0) 6221 56 8815.
E-mail address: klaus.podar@nct-heidelberg.de (K. Podar).
0304-3835/$ - see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.canlet.2013.09.042

compromise, and immunodeciency. Despite unprecedented advances in our understanding of disease pathogenesis and treatment during the last decade, median overall survival of MM
patients remains at 78 years. Therefore, the identication and
validation of targets for novel therapeutic approaches is urgently
needed.
In tumor cells, the nely tuned homeostatic balance between
cell growth and cell death is disturbed. Deregulated expression,
degradation and function of anti-apoptotic and pro-apoptotic
members of the B-cell lymphoma-2 (Bcl-2) family have been

F. Fan et al. / Cancer Letters 343 (2014) 286294

implicated in the development of virtually all hematologic and

solid malignancies [1]. Myeloid cell leukemia-1 (MCL-1) gene, located at chromosome 1q21, encodes a strong anti-apoptotic protein of the Bcl-2 family. Functionally, Mcl-1 is involved in cell
homeostasis and differentiation; in the development and maintenance of B and T cells; in the survival of hematopoietic stem cells
[24]; and in the maintenance of long-lived plasma cells in their
BM niche [5]. Structurally, Mcl-1 is unique among the Bcl-2 family. As other anti-apoptotic Bcl-2 family proteins, Mcl-1 contains
Bcl-2 homology regions (BH 14 domains) and a transmembrane
domain, which confer the ability to heterodimerize with other
family members (e.g. pro-apoptotic proteins Bak and Bax) or to
bind to membranes (e.g. cell membrane, mitochondrial membrane, nuclear envelope). Mcl-1 differs from its pro-survival
family members in its larger size of 350 residues due to the
N-terminal presence of two weak and two strong polypeptide
sequences enriched in proline (P), glutamic acid (E), serine (S)
and threonine (T) (PEST domain). The PEST domain is rich in
regulatory residues and motifs including sites for cleavage,
ubiquitination and phosphorylation. Dependent on cellular
conditions such as the presence of growth factors and cytokines
or the interaction with other cells, these residues and motifs are
responsible for Mcl-1 stability, localization, dimerization, and
thereby for its function [6].
Caspase-induced Mcl-1 cleavage occurs at two distinct, highly
conserved sites, Asp127 and Asp157. Mcl-1 cleavage at Asp127 results in the generation of two fragments, 28 kDa Mcl-1128350 and
17 kDa Mcl-11127; whereas cleavage at Asp157 results in 23 kDa
Mcl-1158350 and 21 kDa Mcl-11157 cleaved products [79]. The
two C-terminal Mcl-1-cleaved products, Mcl-1128350 and Mcl1158350, have lost the N-terminal BH4 domain and are structurally
similar to Bax. Besides impairing the ability of Mcl-1 to sequester
pro-apoptotic proteins (e.g. Bax, Bak and Bim) and thereby to promote survival, caspase-induced cleavage converts Mcl-1 to a proapoptotic protein [911]. Taken together, Mcl-1 cleavage is an
important process by which cell viability may be rapidly regulated.
Induction of Mcl-1 cleavage is therefore a promising novel strategy
for cancer therapy [12].
In MM, Mcl-1 plays a key role in tumor cell survival and drug
resistance [1317]. Indeed, Mcl-1 is among the signicantly
up-regulated molecules in MM versus normal plasma cells in an
identical genotypic background [18]. Increasing data emerge on
regulation and mechanistic sequelae of Mcl-1 in MM [19].
Based on these ndings, Mcl-1 becomes a promising target for
MM therapy. Preclinical approaches to deplete Mcl-1 in MM
cells include targeting the ubiquitin-dependent pathway
(e.g. deubiquitinase USP9X inhibitors) [20]; BH-3 mimetics (e.g.
obatoclax, GX015-070) [21]; the galectin-3 antagonist GCS-100
[22]; and overexpression of microRNA-29b [23,24]. Several
ongoing clinical trials evaluate the activity of obatoclax in a variety
of solid and hematologic malignancies including MM (http://
www.clinicaltrials.gov/).
Our own and other previous studies have shown that
bortezomib-induced generation of Mcl-1 fragment Mcl-1128350,
but not Mcl-11127, triggers Bax-dependent apoptosis in MM cells
[11,25,26]. However, exact molecular mechanisms by which proapoptotic Mcl-1128350 triggers MM cell death are unknown.
Here, we show that a number of preclinical and clinical compounds induce generation of Mcl-1128350 in MM cells. Functionally, the particular novelty of our data is the demonstration of a
new molecular mechanism by which pro-apoptotic Mcl-1128350
triggers upregulation of c-Jun mRNA and protein levels, followed
by increased AP-1 activity in MM cells. Importantly, our data suggest that this mechanism is operative not only in MM but also in
other hematologic and solid malignancies, which express high levels of Mcl-1, glioblastoma in particular.

287

2. Materials and methods

2.1. Materials
Bortezomib, ixazomib (MLN9708), carlzomib, and adaphostin (NSC 680410),
the adamatyl ester analogue of the tyrophostin AG957 were purchased from
Selleck Chemicals; staurosporine was from Sigma; MG-132 was from Merck.
Antibodies against human Mcl-1 (S-19), c-Jun (H-79), extracellular signalregulated kinase 2 (ERK2) were from Santa Cruz Biotechnology; antibodies against
histone deacetylase 1 (HDAC1), and PARP were from Cell Signaling Technology;
anti-a-tubulin was from Sigma; anti-murine Mcl-1 was from Rockland Immunochemicals; Alexa Fluor 488 goat anti-rabbit IgG was from Molecular Probes. The
Mcl-1 (S-19) antibody, raised against a peptide mapping amino acid residues
121138 of human Mcl-1, is able to detect full length Mcl-1, as well as the
fragments Mcl-11157 and Mcl-1128350 [7,10].

2.2. Cell culture

All human MM cell lines as well as K-562, HT-29 and ACHN cells were cultured
in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (PAA Laboratories), 1% penicillin/streptomycin and 2 mM
L-glutamine (all from Gibco). T98G, U-87 MG, Hela and 786-0 cells were cultured
in Dulbeccos modied Eagles medium (DMEM) (Gibco) supplemented with 10%
heat-inactivated FBS, 1% penicillin/streptomycin and 2 mM L-glutamine.
MEF cell lines Mcl-1wt/wt and Mcl-1D/null were generated by SV40 large T transformation followed by Tat-Cre-mediated deletion. Single cell clones were selected
and then grown in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 2-mercaptoethanol (Sigma) and Minimum
Essential Medium (MEM) non-essential amino acids (Gibco) from early passages.
Both Mcl-1wt/wt and Mcl-1D/null were extensively characterized as being hypersensitive to various death stimuli with restorable resistance upon re-expression of
Mcl-1 [4].

2.3. Plasmids and cell transfection

Human and murine Mcl-1 expression plasmids pcDNA3.1-hMcl-1 and
pcDNA3-HA-mMcl-1 [27], 3AP-1 reporter [28] were obtained from Addgene.
Mcl-1128350 was a kind gift from Dr. Graham Packman (University of Southampton,
UK).
For the generation of Mcl-1 D127A and Mcl-1 D157A mutants, Asp127 and
Asp157 were individually mutated to alanines using GeneArt Site-Directed Mutagenesis System (Invitrogen) with pcDNA3.1-hMcl-1 as the template following the
manufacturers instructions. The primers used for Mcl-1 D127A were 50 -GAAGAGGAGCTGGCCGGGTACGAGCCG-30 and 50 -CGGCTCGTA CCCGGCCAGCTCCTCTTC-30 .
The primers used for Mcl-1 D157A were 50 -ATAACACCAGTACGGCCGGGTCACTACCCTC-30 and 50 -GAGGGTAGTGACCCGGCCGTACTGGTGTTAT-30 . The mutated
Mcl-1 constructs were veried by sequencing.
MM.1S cells were transiently transfected with indicated plasmids using the
Amaxa Cell Line Nucleofector Kit V (Lonza) according to the manufacturers instructions. Mcl-1wt/wt and Mcl-1D/null MEFs were transiently transfected with indicated
plasmids using Lipofectamine 2000 (Invitrogen).

2.4. Cell lysis, fractionation and western blot analysis

Whole cell lysates were prepared in Frackeltons lysis buffer (10 mM TrisCl,
50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, pH 7.05) supplied
with Halt Protease and Phosphatase Inhibitor Cocktail (Pierce). Extracts of cytoplasmic and nuclear fractions were prepared using Nuclear Extract Kit (Active Motif)
according to the manufacturers instructions. Western blot analysis was performed
as previously described [11].

2.5. Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was puried with RNeasy Mini Kit (Qiagen) according to the manufacturers instructions, then reverse transcribed and synthesized to complementary
DNA (cDNA) using Omniscript reverse transcriptase (Qiagen). PCR amplication
was performed using Taq DNA polymerase (Qiagen). The primers used to amplify
human Mcl-1 were 50 -ATCTCTCGGTACCTTCGGGAGC-30 and 50 -CCTGATGCCACCTTCTAGGTCC-30 . The primers for human c-Jun were 50 -ACTCGGACCTCCTCACCTCG-30
and 50 -TGTTTAAGCTGTGCCACCTGTT-30 . The primers for human 18S rRNA were
50 -CAGCCACCCGAGATTGAGCA-30 and 50 -TAGTAGCGACGGGCGGTGTG-30 . cDNA
samples were also analyzed by quantitative real-time PCR using QuantiFast SYBR
Green PCR kit (Qiagen) with primers: 50 -GAGACCTTACGACGGGTT-30 and
50 -TTTGATGTCCAGTTTCCG-30 for human Mcl-1, 50 -CTCCAAGTGCCGAAAAAGGAAG-30
and 50 -CACCTGTTCCCTGAGCATGTTG-30 for human c-Jun, 50 -TGCTGTCTCCATGTTTGATGTATCT-30 and 50 -TCTCTGCTCCCCACCTCTAAGT-30 for human b-2-microglobulin.

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2.6. Luciferase assay

Cells were transiently transfected with 3  AP-1 reporter or pGL2-basic
vector, together with pRL-CMV Renilla luciferase reporter as an internal control.
In some experiments, cells were co-transfected with plasmids expressing
c-Jun, Mcl-1128350, Mcl-1 wild-type or its mutants. After the indicated treatment,
cells lysates were prepared and luciferase activity was measured using a DualLuciferase Reporter Assay System (Promega) with a single tube luminometer
(Berthold Detection System) according to the manufacturers instructions.
The rey luciferase relative light units (RLU) were normalized to Renilla
luciferase RLU.
2.7. Immunouorescence
MM.1S cells treated with or without bortezomib were collected and 3  104
cells in 100 ll medium were attached on glass slides by centrifugation at
300 rpm for 5 min using a Shandon Cytospin Cytocentrifuge (Thermo Scientic).
Then cells were xed with 4% paraformaldehyde in phosphate-buffered saline
(PBS), permeabilized with 0.1% Triton X-100 in PBS for 5 min and incubated with

primary and secondary antibodies, sequentially. Slides were counterstained with

VECTASHIELD HardFSet Mounting Medium with DAPI (Vector Laboratories). Immunouorescence was observed using a Zeiss Axiophot uorescence microscope with
a 40/0.75 numerical aperture objective. Images were captured by a CCD camera
with Axiovision software (Carl Zeiss Microscopy). After data acquisition, images
were processed using ImageJ software (NIH).

2.8. Apoptosis assay

Cells were treated as indicated, then washed with PBS and co-stained with FITClabeled Annexin V and propidium iodide (PI) using the FITC Annexin V Apoptosis
Detection Kit I (BD Pharmingen) following the manufacturers instructions. Apoptosis was analyzed on a FACSCanto II ow cytometer (BD Biosciences).

2.9. Statistics
Statistical analysis was done using the Students t-test. P-values are indicated by
asterisks (P < 0.01, P < 0.05).

Fig. 1. Drug-induced generation of Mcl-1 fragment Mcl-1128350 correlates with c-Jun upregulation. (a) mRNA levels of Mcl-1 in MM cells. Total RNA was prepared from indicated
human MM cell lines and used in reverse transcription-polymerase chain reaction (RT-PCR) to determine the relative expression level of Mcl-1 with 18S rRNA as a control. (b)
Protein levels of Mcl-1 in MM cells. Cell lysates were prepared from the indicated MM cells. Aliquots of cell extracts (40 lg) were used in western blot with antibodies against
Mcl-1. Tubulin served as a loading control. (c) MM.1S cells were treated with 5 lM melphalan (Mel), 100 nM doxorubicin (Dox), 100 lM thalidomide (Thal), 5 lM adaphostin
(Ada), 500 nM staurosporine (STS), 10 nM bortezomib (Bort), or left untreated (control, Ctrl). After 24 h, cell lysate was immunoblotted with indicated antibodies. Arrow
indicates the position of the 28 kDa Mcl-1 fragment. (d) Bortezomib-induced generation of Mcl-1128350 is associated with c-Jun upregulation in different MM cell lines.
Indicated MM cell lines were treated with or without bortezomib for 24 h, followed by immunoblot analysis of the lysates for the expression of Mcl-1 and c-Jun. Tubulin was
used as a loading control. (e) Induction of c-Jun mRNA expression by drug treatment. Total RNA was puried from MM.1S cells treated as described in (c). The relative
expression levels of Mcl-1 and c-Jun were analyzed by RT-PCR with 18S rRNA as a control. For Mcl-1, the 444 bp product corresponds to the full-length transcript, while the
smaller product (196 bp) represents the short alternative splice variant [48]. (f) Quantitative PCR (qPCR) analysis of Mcl-1 and c-Jun mRNA levels in samples from (e). Beta-2microglobulin served as an endogenous control. The symbols of and indicate P < 0.05 and P < 0.01, respectively. (g) Other proteasome inhibitors that trigger Mcl-1128350
generation and c-Jun upregulation. MM.1S cells were treated with carlzomib (Carf), ixazomib (Ixaz), MG-132 (MG), or left untreated. After 24 h, total cell lysate was
immunoblotted with Mcl-1, c-Jun and Tubulin.

F. Fan et al. / Cancer Letters 343 (2014) 286294

3. Results
3.1. Drug-induced generation of Mcl-1128350 correlates with c-Jun
upregulation
Numerous preclinical studies have indicated the requirement
of anti-apoptotic protein Mcl-1 for MM cell survival. Here, we
examined the expression of Mcl-1 in six human MM cell lines
(RPMI 8226, DOX40, U266, OPM2, MM.1S, MM.1R) and conrmed
high levels of Mcl-1 mRNA (Fig. 1a) and protein (Fig. 1b) in MM
cells. Several anti-MM drugs have been reported to downregulate
expression levels of full length Mcl-1 via a ubiquitin-dependent
pathway, thereby inducing apoptosis. Our own and other previous
data have demonstrated the ability of bortezomib to induce
caspase-dependent generation of pro-apoptotic Mcl-1128350 fragment [11,25]. To identify additional compounds which are able
to trigger generation of Mcl-1128350 cleaved protein in MM cells,
we tested a number of preclinical and clinical compounds including melphalan, doxorubicin, thalidomide, adaphostin, bortezomib,
and staurosporine. All drugs investigated induced MM cell

289

apoptosis (data not shown). However, generation of Mcl-1128350

was only observed upon treatment with bortezomib, staurosporine
and adaphostin (Fig. 1c).
We next sought to determine the downstream molecular sequelae which trigger anti-MM effects of Mcl-1128350. Based on our
previous studies which have shown that upregulation of c-Jun in
MM cells inhibits proliferation and induces apoptosis, we investigated whether generation of Mcl-1128350 is associated with the
pro-apoptotic function of c-Jun [29]. Indeed, drug-induced generation of Mcl-1128350 correlated with c-Jun protein upregulation in
MM cell lines MM.1S (Fig. 1c), RPMI 8226, DOX40, U266, OPM2,
and MM.1R (Fig. 1d). Consistent with these data, RT-PCR analysis
showed increased levels of c-Jun mRNA in MM cells treated with
adaphostin, staurosporine and bortezomib, but not melphalan,
doxorubicin and thalidomide (Fig. 1e). These results were further
conrmed by quantitative real-time PCR (Fig. 1f). Interestingly,
similar to bortezomib, the next generation proteasome-inhibitors
carlzomib and ixazomib, as well as the investigational proteasome inhibitor MG-132 also triggered generation of Mcl-1128350
and upregulation of c-Jun protein (Fig. 1g).

Fig. 2. Mcl-1128350 triggers c-Jun upregulation and AP-1 transcriptional activity. (a) Induction of c-Jun mRNA by Mcl-1128350 expression. MM.1S cells were transfected with
plasmids encoding empty vector (pcDNA3), wild-type Mcl-1 (Mcl-1wt), the Mcl-1 fragment (Mcl-1128350) or c-Jun. After 24 h, total RNA was puried and reverse transcribed
to cDNA. Mcl-1 and c-Jun were detected by PCR, with 18S rRNA as a control. Note that the primer pair for Mcl-1 was able to amplify the cDNA region encoding the
Mcl-1128350. (b) Mcl-1128350 triggers upregulation of c-Jun protein levels. MM.1S cells were transfected with pcDNA3, Mcl-1wt or Mcl-1128350. Cell lysates were
immunoblotted with antibodies against Mcl-1 and c-Jun. ERK2 was used as a loading control (upper panel). Lower panel: densitometric analysis of Mcl-1 full length relative
to ERK2 from three independent experiments. (c) MM.1S cells were transfected with empty vector or c-Jun. Cell lysates were immunoblotted with antibodies against Mcl-1
and c-Jun. ERK2 was used as a loading control. (d) Accumulation of Mcl-1 fragment in the nucleus is associated with nuclear c-Jun upregulation. MM.1S cells were transfected
with pcDNA3 or Mcl-1128350. Cytoplasmic and nuclear fractions were prepared and immunoblotted with antibodies as indicated. HDAC1 and Tubulin were used as purity
controls of nuclear (HDAC1) and cytoplasmic fractions (Tubulin). (e) Mcl-1128350, but not full length Mcl-1, induces AP-1 activity. MM.1S cells were transiently transfected
with indicated constructs. Transfection mixes also contained a plasmid encoding Renilla luciferase for normalization. After 18 h, the whole cell lysate was analyzed by
dual-luciferase reporter assay. Relative rey luciferase reporter activity was determined as the percentage of the Renilla luciferase activity. The results were presented as
mean standard deviation (SD) of triplicate samples. Insert panel: positive control for the AP-1 reporter luciferase assay using c-Jun expression plasmid.

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F. Fan et al. / Cancer Letters 343 (2014) 286294

3.2. Mcl-1128350. triggers c-Jun upregulation and AP-1 transcriptional

activity
In order to investigate the potential functional interrelation between Mcl-1128350 and c-Jun upregulation, we transfected MM.1S
cells with plasmids carrying Mcl-1wt, Mcl-1128350 or control vector
and measured c-Jun expression. The results showed increased levels
of c-Jun mRNA (Fig. 2a) as well as c-Jun protein (Fig. 2b) in
Mcl-1128350-expressing MM cells. In contrast, overexpression of
c-Jun did not induce the generation of Mcl-1 fragment Mcl-1128350
(Fig. 2c). Interestingly, we found that overexpressed Mcl-1128350,
but not full-length Mcl-1, accumulated in the nuclear fraction
(Fig. 2d). These data suggest a role for Mcl-1128350 in the regulation
of c-Jun transcription. Consequently, overexpression of Mcl-1128350,
but not Mcl-1wt, enhanced the transcriptional activity of c-Jun in MM
cells (Fig. 2e). AP-1 activity triggered by transfection of MM cells
with c-Jun expression plasmid served as a positive control (Fig. 2e,
insert). Taken together, these data demonstrate for the rst time a
regulatory role of Mcl-1 128350 in AP-1 transcription and activity.
3.3. Drug-induced generation of Mcl-1128350 triggers pro-apoptotic
c-Jun activity
Similar to exogenous Mcl-1128350, drug-induced Mcl-1 fragment Mcl-1128350 predominantly localized within the nucleus
(Fig. 3a and b) and triggered transcriptional activity of c-Jun
(Fig. 3c).
To conrm a specic role of the Mcl-1 fragment Mcl-1128350 in
the induction of c-Jun transcriptional activity, we generated constructs carrying Mcl-1 mutants Mcl-1 D127A and D157A. Specically, the highly conserved Mcl-1 caspase-cleavage sites Asp127

and Asp157 were individually mutated to alanines by site-directed

mutagenesis with the primers indicated (Fig. 4a). Consistent with
our hypothesis, bortezomib-triggered induction of c-Jun activity
was signicantly inhibited in MM cells transfected with Mcl-1
D127A, but not with Mcl-1 D157A (Fig. 4b). Consequently, bortezomib-induced MM cell apoptosis was signicantly decreased in Mcl1 D127A-overexpressing MM cells (Fig. 4c). Taken together, these
results demonstrate that drug-induced generation of Mcl-1128350
enhances pro-apoptotic c-Jun expression and activity.
3.4. Drug-induced generation of a pro-apoptotic Mcl-1 fragment might
also be a novel therapeutic approach in other tumor entities
To further verify our ndings, we next utilized wild-type (Mcl1wt/wt) and Mcl-1-null (Mcl-1D/null) murine embryonic broblasts
(MEFs) [4]. Indeed, bortezomib triggered c-Jun upregulation in
Mcl-1wt/wt MEFs, but not in Mcl-1D/null MEFs (Fig. 5a). Importantly,
re-expression of wild-type murine Mcl-1 (mMcl-1wt) in Mcl-1D/null
MEFs restored the effect of bortezomib on c-Jun expression
(Fig. 5b). Similar to MM cells, bortezomib-induced Mcl-1128350
was predominantly localized within the nuclear fraction (data
not shown). Consistently, treatment with bortezomib signicantly
elevated AP-1 transcriptional activity in Mcl-1wt/wt, but not Mcl1D/null, MEFs (Fig. 5c). Moreover, when Mcl-1D/null MEFs were
transfected with a plasmid carrying mMcl-1wt, bortezomib-induced upregulation of AP-1 activity was rescued (Fig. 5d). The
presence of the Mcl-1 fragment correlated with induction of MEF
apoptosis (data not shown).
Mcl-1 overexpression has been extensively reported in hematologic cancers including MM, where it is also associated with poor
prognosis (Suppl. Fig. 1). However, until recently its relevance in

Fig. 3. Drug-induced accumulation of Mcl-1 fragment in the nucleus is associated with nuclear c-Jun upregulation and induction of AP-1 activity. (a) Drug-induced accumulation of
Mcl-1 fragment in the nuclear fractions is associated with nuclear c-Jun upregulation. MM.1S cells were treated with bortezomib (Bort), staurosporine (STS), or left untreated.
Cytoplasmic and nuclear fractions were prepared and analyzed by western blot with indicated antibodies. HDAC1 and Tubulin were used as purity controls of nuclear
(HDAC1) and cytoplasmic fractions (Tubulin). (b) Drug-induced accumulation of Mcl-1 fragment in the nucleus. MM.1S cells were treated with bortezomib (Bort), xed, and
stained with anti-human Mcl-1 antibody (S-19) (green). To visualize the nuclei, cells were counterstained with DAPI (blue). The immunouorescence staining was examined
by uorescence microscopy. Non-treated MM.1S cells were used as control (Ctrl). (c) Induction of AP-1 activity by drug treatment. MM.1S cells were transiently transfected
with 7 lg 3 AP-1 reporter and 1 lg pRL-CMV Renilla luciferase vector, then treated with 5 lM melphalan (Mel), 100 nM doxorubicin (Dox), 100 lM thalidomide (Thal),
500 nM staurosporine (STS), 10 nM bortezomib (Bort), 20 nM carlzomib (Carf), 40 nM ixazomib (Ixaz), 200 nM MG-132 (MG) or left untreated (control, Ctrl). Cell lysates
were prepared, and luciferase activity was measured by dual-luciferase reporter assay. The fold change of luciferase activity relative to control cells is shown as mean SD
from three independent experiments. The symbols of and indicate P < 0.05 and P < 0.01, respectively. (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.)

F. Fan et al. / Cancer Letters 343 (2014) 286294

291

studies, of intense overexpression in patients affected by glioblastoma (Fig. 5e, and data not shown). We then asked whether treatment with the aforementioned drugs was able to trigger Mcl-1
cleavage in tumor cell lines other than MM overexpressing Mcl-1. Indeed, our results showed that drug treatment induced signicant
generation of Mcl-1 fragment in a variety of human tumor cell lines
(chronic myelogenous leukemia, cervix adenocarcinoma, colorectal
adenocarcinoma, and renal adenocarcinoma (Suppl. Fig. 2)), glioblastoma in particular (Fig. 5f). Consistent with our data, a previous
study showed that bortezomib induces cell death in several glioblastoma cell lines [33]. Taken together, these data suggest that drug-induced generation of a pro-apoptotic Mcl-1 fragment might also be a
novel therapeutic approach in tumor entities other than MM, e.g.
glioblastoma.

4. Discussion

Fig. 4. Mutation of the Mcl-1 residue Aspartate (Asp) 127, but not Asp157, signicantly
decreases bortezomib-induced c-Jun upregulation and MM cell apoptosis. (a) Schematic
representation of wild-type (wt) Mcl-1 and the mutants D127A, D157A. Numbers
indicate amino-acid residues. The highly conserved Mcl-1 caspase-cleavage sites
Asp127 and Asp157 were individually mutated to alanine by site-directed mutagenesis with the primers indicated. Mutations were veried by sequencing. (b) Druginduced upregulation of AP-1 activity is signicantly decreased after introduction of a
point mutation into Mcl-1 caspase-cleavage site Asp127. MM.1S cells were transfected with 4 lg Mcl-1 wt, D127A or D157A, 4 lg AP-1 reporter and 0.8 lg Renilla
luciferase vector, and then treated with bortezomib or left untreated. Whole cell
lysates were analyzed by dual-luciferase reporter assay. The relative fold change of
luciferase activity is shown as mean SD from three independent experiments. The
luciferase activity in Mcl-1 wt group without drug treatment was set to 1. Indicates
P < 0.05 by Students t-test. (c) Drug-triggered MM cell apoptosis is signicantly
reduced after introduction of a point mutation into Mcl-1 caspase-cleavage site
Asp127. MM.1S cells were transfected with Mcl-1 wt, D127A or D157A, and then
treated with or without bortezomib. Cell apoptosis was analyzed by Annexin V/PI
staining. The percentage of cells staining positive for Annexin V is shown as mean SD
of three independent experiments. Indicates P < 0.01.

solid cancers has been questioned. Indeed, in neuroblastoma, the

role of Mcl-1 as an anti-apoptotic cancer gene has been challenged
[30]. Notwithstanding, a recent survey of copy number aberrations
in a panel of 3,131 cancer specimens belonging to 26 histological
types has highlighted a focal region on chromosome 1q21.2 as one
of the most frequently amplied regions of the cancer genome
[31]. Additional functional data demonstrated that Mcl-1 represents
the most cogent oncogenic candidate within this amplicon. Most
importantly, Mcl-1 amplications were shared among several cancer types including epithelial cancers, suggesting that Mcl-1 deregulation exerts a major role not only in hematologic cancers, but
also in solid tumors. In line with these results, a recent shRNA screen
in glioma has suggested a prominent role for Mcl-1 in protecting
malignant glioma cells from apoptosis [32]. Thus, we conducted an
extensive survey of published datasets to uncover tumor types demonstrating a particularly strong overexpression of Mcl-1 in cancer
tissues when compared with their normal counterparts. Among
other tumor types, we identied a pattern, consistent across several

Mcl-1 is required for long-term survival of plasma cells and intimately involved in MM pathogenesis and drug resistance. Based on
these ndings, Mcl-1 represents a promising therapeutic target in
MM [12,34,35].
E3-ubiquitin ligases Mule, b-TrCP, and SCFFBW7, as well as the
deubiquitinase USP9X, are key factors, which regulate Mcl-1 ubiquitination and proteasome-dependent Mcl-1 degradation. Induction of proteasome-dependent Mcl-1 degradation has been
investigated as a potential therapeutic strategy in cancer treatment
including MM. However, mutational inactivation of enzymes associated with proteasomal Mcl-1 degradation occurs in a variety of
malignancies [3639], suggesting that tumor cells develop effective strategies to sustain the increased expression of Mcl-1. Indeed,
a somatic mutation affecting USP9X, with the substitution of a lysine with an asparagine, has been recently reported in MM,
although its functional impact remains to be ascertained [40].
Compounds targeting the proteasome-dependent pathway of
Mcl-1 degradation may not be effective. Therefore, the induction
of non-proteasomal degradation of Mcl-1 may represent an
alternative to be explored for cancer therapy in general, and MM
in particular. Indeed, sequencing results of Mcl-1 in 23 MM cell
lines did not show the existence of point mutations in Mcl-1 caspase-cleavage sites, excluding a potential mechanism of drug resistance in MM (Suppl. Fig. 3).
Using a drug screen to identify Mcl-1128350-generating agents
in MM cells, our results show that, similar to bortezomib [11,25],
staurosporine and adaphostin, as well as next-generation proteasome inhibitors carlzomib and ixazomib, induce Mcl-1128350
generation. One mechanism underlying the observation that proteasome inhibitors induce Mcl-1 fragmentation might be enhanced
stabilization and accumulation of full-length anti-apoptotic Mcl-1
leading to generation of sufcient levels of pro-apoptotic Mcl1128350. Based on these data, we hypothesize that the anti-MM
activity of proteasome inhibitors, at least in part, is mediated via
generation of Mcl-1128350. In ongoing studies, we are utilizing a
large-scale drug screen to identify additional compounds which
are able to induce Mcl-1128350 generation.
Given that drug-induced generation of Mcl-1128350 is a promising novel therapeutic approach in MM therapy, we next sought to
decipher molecular sequelae downstream of Mcl-1128350 which
mediate its pro-apoptotic effect. The pivotal functional role of
subcellular Mcl-1 localization has been emphasized in a number
of recent studies [1,41]. Surprisingly, we observed nuclear accumulation of drug-induced or exogenously transfected Mcl-1128350 in
MM cells. These results suggested a potential regulatory role of
Mcl-1128350 in gene transcription and cell cycle control in MM
cells. Sequence analyses using both the PredictProtein server
(https://www.predictprotein.org/) [42] and YLoc (http://abi.inf.

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F. Fan et al. / Cancer Letters 343 (2014) 286294

Fig. 5. Drug-induced generation of a pro-apoptotic Mcl-1 fragment might also be a novel therapeutic approach in other tumor entities. (a) Bortezomib triggers c-Jun upregulation in
Mcl-1wt/wt MEFs, but not in Mcl-1D/null MEFs. Mcl-1wt/wt and Mcl-1D/null MEFs were cultured in the presence or absence of 10 nM bortezomib for 48 h. (b) Expression of mMcl1wt in Mcl-1D/null MEFs restores bortezomib-triggered c-Jun upregulation. Mcl-1D/null MEFs were transiently transfected with pcDNA3 or pcDNA3-HA-mMcl-1. After 12 h,
cells were treated with or without 10 nM bortezomib for 48 h. Cell lysates were examined by immunoblot analysis with indicated antibodies. Tubulin served as a loading
control (a and b). (c) Bortezomib induces AP-1 activity in Mcl-1wt/wt MEFs, but not in Mcl-1D/null MEFs. Mcl-1wt/wt and Mcl-1D/null MEFs were transfected with AP-1 reporter
and Renilla luciferase vector. (d) Expression of mMcl-1wt in Mcl-1D/null MEFs restores bortezomib-induced AP-1 activity. Mcl-1D/null MEFs were transfected with AP-1 reporter,
Renilla luciferase vector, as well as pcDNA3 or pcDNA3-HA-mMcl-1. Transfections were performed in duplicate in a 12-well plate format. MEFs were treated with or without
10 nM bortezomib for 48 h. Whole cell lysates were used in a dual-luciferase assay, and the fold change of luciferase activity is shown as the mean SD. The relative luciferase
activities without bortezomib treatment were set to 1, respectively. Indicates P < 0.01 (c and d). (e) Upregulation of Mcl-1 in glioblastoma (GBM). Oncomine analysis of
samples isolated from glioblastoma patients shows a signicant upregulation of Mcl-1 expression when compared with expression values of samples isolated from healthy
individuals (based on TCGA publicly available data: 515 patient samples versus 10 healthy controls, Affymetrix U133A array, probe set 200798_x_at). (f) Bortezomib-induced
generation of Mcl-1128350 is associated with c-Jun upregulation in glioblastoma cell lines. T98G and U-87 MG cells were treated with or without bortezomib for 24 h,
followed by immunoblot analysis of cell lysates with antibodies against Mcl-1 and c-Jun. Tubulin was used as a loading control.

uni-tuebingen.de/Services/YLoc/webloc.cgi) [43] showed that full

length Mcl-1 is located in the cytoplasm. Furthermore, the PredictProtein server failed to identify a classical nuclear localization signal (NLS) in Mcl-1 protein sequence, and the NetNES software
(http://www.cbs.dtu.dk/services/NetNES/) [44] failed to nd a
nuclear export signal (NES) in the protein (data not shown).
Therefore, our ongoing studies aim to identify proteins which
interact with Mcl-1128350 to provide the necessary NLS function
and facilitate its nuclear import in MM cells.
Functionally, our present data demonstrate that the generation
of Mcl-1128350 induces marked elevation of c-Jun mRNA and protein levels, as well as enhanced AP-1 reporter activity. Consistent
with these ndings, drug-induced upregulation of pro-apoptotic
c-Jun was blocked after introduction of a point mutation into the
highly conserved Mcl-1 caspase-cleavage site Asp127. In contrast,
introduction of a point mutation into the Mcl-1 cleavage site
Asp157 did not decrease bortezomib-induced c-Jun upregulation,
further emphasizing a key role of Mcl-1128350 in c-Jun transcription. However, the DNAbind web server (http://www.enzim.hu/
~szia/dnabind.html) [45] predicted Mcl-1 as a non DNA-binding
protein, based on its sequence. Thus, future studies will focus to
further explore the functions of Mcl-1 fragment in the nucleus.

Although most studies identied c-Jun as an enhancer of proliferation, a tumor promoter and oncogene, recent studies show that
c-Jun also mediates cell death either by acting as a transcriptional
regulator of survival/death genes (e.g. Fas ligand, DP5, Bim,
p14ARF/p19ARF, Dmp1) and as an inducer of DNA repair responses,
or by triggering caspase-mediated cleavage of numerous molecules
(e.g. fodrin, poly (ADP-ribose) polymerase (PARP), DNA-dependent
protein kinase, and protein kinase C) [46]. More recently, the apoptosis-antagonizing transcription factor (AATF) has been identied
as a nucleolar-conned c-Jun cofactor whose expression levels
and spatial distribution determines the levels of c-Jun-mediated
apoptosis [47]. Potential explanations for its opposing effects include cell type specicity, the availability of external and internal
survival factors, and specic spectra of binding partners [46]. Exact
molecular mechanisms conferring c-Jun-induced cell death in MM
cells are still elusive and under active investigation.
To further support our ndings, we examined effects of Mcl-1
fragment-generating drugs in Mcl-1wt/wt and Mcl-1D/null MEFs
[4]. Our results show that bortezomib signicantly increased
c-Jun protein levels as well as c-Jun transcriptional activity in
Mcl-1wt/wt, but not Mcl-1D/null, MEFs. These data suggest a potential therapeutic role for drug-induced generation of the Mcl-1

F. Fan et al. / Cancer Letters 343 (2014) 286294

293

References

Fig. 6. Drug-induced generation of Mcl-1 fragment Mcl-1128350 triggers MM cell death

via c-Jun upregulation. A variety of compounds induces proteasome-independent
generation of the pro-apoptotic Mcl-1 fragment Mcl-1128350, which triggers c-Jun
upregulation followed by increased AP-1 activity and tumor cell death. Our data
suggest that a currently unknown protein containing an NLS region facilitates
nuclear translocation of the Mcl-1 fragment, and thereby enables Mcl-1128350dependent c-Jun upregulation.

fragment also in settings other than MM. Indeed, recent evidence

from the literature points to a much wider role for Mcl-1 in carcinogenesis, including solid cancers as well as hematologic malignancies. Our own survey on published datasets identied several
cancer types with remarkably high levels of Mcl-1 expression, glioblastoma in particular. Moreover, we found that bortezomib triggered the generation of pro-apoptotic Mcl-1128350 in human
tumor cell lines including glioblastoma, chronic myelogenous leukemia, cervical adenocarcinoma, colorectal adenocarcinoma, and
renal adenocarcinoma.
In summary, the present study conrms Mcl-1 as a promising
target in MM therapy. Specically, our data propose a new
therapeutic approach by which specic compounds induce proteasome-independent generation of the pro-apoptotic Mcl-1 fragment
Mcl-1128350, followed by c-Jun upregulation, increased AP-1 activity, and the induction of MM cell death (Fig. 6). Finally, our results
suggest that this mechanism may be operative not only in MM, but
also in other malignancies.
Conict of Interest
All authors declare no conicts of interest.
Acknowledgements
We thank Dr. G. Packham (University of Southampton School of
Medicine, Southampton General Hospital, Southampton, UK) for
kindly providing Mcl-1128350 plasmid; and Drs. M. Trier and D.
Bohmann for kindly providing c-Jun plasmid. K. Podar is a recipient
of a B. Braun Stiftungs-Grant.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.canlet.2013.
09.042.

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