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Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
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
287
288
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.
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
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.
290
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.)
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.
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.
292
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.
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
293
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