Mcl-1 ubiquitination and destruction.

Abstract

Loss of the Fbw7 tumor suppressor is common in diverse human cancer types, including T-Cell Acute Lymphoblastic Leukemia (T-ALL), although the mechanistic basis of its anti-oncogenic activity remains largely unclear. We recently reported that SCFFbw7 regulates cellular apoptosis by controlling the ubiquitination and destruction of the pro-survival protein, Mcl-1, in a GSK3 phosphorylation-dependent manner. We found that human T-ALL cell lines displayed a close relationship between Fbw7 loss and Mcl-1 overexpression. More interestingly, T-ALL cell lines that are deficient in Fbw7 are particularly sensitive to sorafenib, a multi-kinase inhibitor that has been demonstrated to reduce Mcl-1 expression through an unknown mechanism. On the other hand, Fbw7-deficient T-ALL cell lines are much more resistant to the Bcl-2 antagonist, ABT-737. Furthermore, reconstitution of Fbw7 or depletion of Mcl-1 in Fbw7-deficient cells restores ABT-737 sensitivity, suggesting that elevated Mcl-1 expression is important for Fbw7-deficient cells to evade apoptosis. Therefore, our work provides a novel molecular mechanism for the tumor suppression function of Fbw7. Furthermore, it provides the rationale for targeted usage of Mcl-1 antagonists to treat Fbw7-deficient T-ALL patients.

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Oncotarget, March, Vol.2, No 3
Oncotarget 2011; 2: 239 - 244www.impactjournals.com/oncotarget 239
Mcl-1 Ubiquitination and Destruction
Hiroyuki Inuzuka, Hidefumi Fukushima, Shavali Shaik, Pengda Liu, Alan W. Lau
and Wenyi Wei
* Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
Correspondence to: Wenyi Wei, email: wwei2@bidmc.harvard.edu
Keywords: Ubiquitination, SCF, Fbw7, GSK3, Mcl-1, Apoptosis, Tumor Suppressor, Phosphorylation, Cell Cycle
Received: March 18, 2011, Accepted: March 18, 2011, Published: March 19, 2011
Copyright: © Inuzuka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
AbstrAct:
Loss of the Fbw7 tumor suppressor is common in diverse human cancer types,
including T-Cell Acute Lymphoblastic Leukemia (T-ALL), although the mechanistic
basis of its anti-oncogenic activity remains largely unclear. We recently reported
that SCFFbw7 regulates cellular apoptosis by controlling the ubiquitination and
destruction of the pro-survival protein, Mcl-1, in a GSK3 phosphorylation-dependent
manner. We found that human T-ALL cell lines displayed a close relationship
between Fbw7 loss and Mcl-1 overexpression. More interestingly, T-ALL cell lines
that are deficient in Fbw7 are particularly sensitive to sorafenib, a multi-kinase
inhibitor that has been demonstrated to reduce Mcl-1 expression through an
unknown mechanism. On the other hand, Fbw7-deficient T-ALL cell lines are much
more resistant to the Bcl-2 antagonist, ABT-737. Furthermore, reconstitution of
Fbw7 or depletion of Mcl-1 in Fbw7-deficient cells restores ABT-737 sensitivity,
suggesting that elevated Mcl-1 expression is important for Fbw7-deficient cells to
evade apoptosis. Therefore, our work provides a novel molecular mechanism for
the tumor suppression function of Fbw7. Furthermore, it provides the rationale
for targeted usage of Mcl-1 antagonists to treat Fbw7-deficient T-ALL patients.
INtrODUctION
Dysregulated cell cycle progression leads to
uneven distribution of the genetic information between
the two daughter cells, which contributes to genomic
instability and ultimately, cancer development. Recent
work established that two related, multi-component
E3 ubiquitin ligase enzymes, the Anaphase Promoting
Complex (APC) and the Skp1-Cullin1-F-box complex
(SCF), are the major driving forces governing proper cell
cycle progression [1-4]. APC is active from the late G2
phase to mid-G1 phase, and is responsible for degradation
of mitotic cyclins, securin and geminin [5, 6]. On the
other hand, SCF is thought to be active from the late G1
phase until the G2 phase and mediates the ubiquitination
and destruction of G1 cyclins and Cdk inhibitors [1, 7].
SCF consists of the adaptor protein Skp1, the scaffold
protein Cul1, the ring-finger protein Rbx1, as well as
a variable component that is responsible for substrate
recognition known as the F-box protein. The human
genome encodes 68 putative F-box proteins, thereby
providing sufficient flexibility for substrate specificity
[8]. Most of the physiological functions of these putative
F-box proteins remain unknown. The well-characterized
F-box proteins include Skp2, Cdc4/Fbw7, and β-TRCP1,
which targets p27 [9], cyclin E [10], and Cdc25A [11],
respectively, for ubiquitination and degradation. In all
cases, proper phosphorylation of the substrate is required
for its interaction with the F-box proteins.
Fbw7 Is A tUmOr sUppressOr
Loss of Fbw7 is frequently observed in various types
of tumors including breast cancer, colon cancer [12] and
T-cell acute lymphoblastic leukemia (T-ALL) [13]. It has
been documented that tissue-specific deletion of Fbw7 in
mouse T cells results in the development of T-ALL [14-
16], suggesting that Fbw7 is a novel tumor suppressor in
T-ALL. However, the exact molecular mechanisms by
which Fbw7 exerts its anti-tumor activity are still unknown
[4]. We previously discovered that Fbw7 regulates
the degradation of c-Jun in a GSK3 phosphorylation-
dependent manner [17]. Our work assigned a biological

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significance to the v-Jun S243F point mutation and also
underscored the importance of Fbw7 in tumor suppression
[17]. In addition to the turnover of cyclin E [10] and c-Jun,
Fbw7 is also involved in the degradation of c-Myc [18,
19], and the Notch-1 protein [20] (Figure 1), all of which
have been reported to possess oncogenic functions and are
frequently found to be overexpressed in various human
cancers, including leukemia. Consistent with frequent loss
of Fbw7, overexpression of c-Myc, c-Jun and Notch-1
is closely associated with the development of T-ALL.
Besides accelerating cell growth [21], overexpression
of either c-Jun, c-Myc or Notch-1 results in cell death
through upregulation of the pro-apoptotic protein Bim-
1 [22]. However, despite the ever-growing list of Fbw7
ubiquitin substrates (Figure 1), it remains unclear how
Fbw7-deficient cells evade cell death in the setting of
upregulated c-Jun, c-Myc or Notch-1 (Figure 2A).
the mcl-1 ONcOprOteIN Is FOUND
tO be FreqUeNtly OverexpresseD
IN leUkemIA
Expression of the anti-apoptotic protein Mcl-1 is
frequently elevated in various human tumors including
leukemia, but the underlying mechanisms causing its
elevation are not fully understood [23, 24]. Mcl-1 is a
pro-survival member of the Bcl-2 family of proteins,
which can suppress apoptosis by interacting with and
suppressing the activities of pro-apoptotic proteins
including Bim, Bax and Bak [25]. However, unlike other
Bcl-2 family members, the Mcl-1 protein is extremely
unstable, having a very short half-life [26]. The rapid
induction and destruction of Mcl-1 has been proposed
as a molecular mechanism for cells to switch into either
the survival or apoptotic pathways in response to various
stresses [27, 28]. Although GSK3 phosphorylation is
reported to regulate Mcl-1 stability directly [26], little is
known about the upstream E3 ubiquitin ligase that targets
phosphorylated Mcl-1 for destruction. As illustrated in
Figure 2B, we and others recently reported that Fbw7
targets Mcl-1 for ubiquitination and destruction in a
GSK3-dependent manner [29, 30]. Therefore, our studies
suggest that the simultaneous elevation of the pro-survival
factor Mcl-1 provides a protection mechanism allowing
Fbw7-deficient cells to evade apoptosis, thus providing
a novel molecular mechanism for the tumor suppression
function of Fbw7 (Figure 2B). Moreover, Mcl-1 has been
demonstrated to play a key role in regulating the cellular
apoptosis of T cells, but not other tissue types such as
liver cells [27, 31]. Therefore, our studies also provide
the possible mechanism for why loss of Fbw7 is very
frequently observed in T-ALL patients.
Figure 1: schematic illustration of the
scFFbw7 e3 ubiquitin ligase complex and a
list of its identified downstream ubiquitin
substrates.
Cul1
Skp1
Rbx1
Fbw7
E2
PP
Substrates
Ub
Reported Fbw7
Substrates
Cyclin E
c-Myc
Notch1
c-Jun
Presenilin
SREBP1
mTOR
c-Myb
KLF5
Mcl-1
Ub
Ub
Ub
c-Myc
c-Jun
Notch1
Cell Death
Pro-apoptotic
Signal
c-Myc
c-Jun
Mcl-1
Notch1
Tumorigenesis
?
Fbw7 Fbw7
Cell Growth
Cell Death
Pro-apoptotic
Signal
Cell Growth
AB
Figure 2: Fbw7 participates in the regulation of cellular apoptosis by targeting the pro-survival factor mcl-1 for
ubiquitination and destruction.
A. Loss of Fbw7 leads to the elevated expression of c-Myc, c-Jun, and the Notch-1 protein, all of which possess oncogenic functions and are
frequently found to be overexpressed in various human cancers, including leukemia. Besides promoting cell growth, it has been shown that
overexpression of either c-Jun, c-Myc or Notch-1 provokes cellular apoptosis. However, it remains unclear how Fbw7-deficient cells evade
programmed cell death in the setting of upregulated c-Jun, c-Myc or Notch-1.
b. We recently reported that loss of Fbw7 also leads to a significant elevation in Mcl-1 expression, which suppresses the induction of apoptosis
by inactivating the pro-apoptotic function of many BH3 only proteins including Bim, Bax and Bak

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the
cells Are “ADDIcteD” tO hIgh
expressION levels OF mcl-1
Fbw7-DeFIcIeNt t-All
Our studies for the first time provide firm
experimental evidence for a potential role for the Fbw7
tumor suppressor in the modulation of the apoptotic
pathway by governing Mcl-1 ubiquitination and
destruction. Therefore, loss of Fbw7 not only provides a
growth advantage by upregulating the c-Jun and c-Myc
oncoproteins, but also leads to elevated Mcl-1 expression
as a protection mechanism allowing Fbw7-deficient cells
to evade possible apoptosis induced by high c-Jun and
c-Myc expression (Figure 2B). However, this intricate
balance to evade apoptosis seems to depend on high levels
of the Mcl-1 oncoprotein. As a result, compared to WT-
Fbw7 cells, Fbw7-deficient T-ALL cells are much more
sensitive to the Mcl-1 inhibitor, sorafenib (Figure 3).
Sorafenib is a multi-kinase inhibitor reported to suppress
B-Raf, PDGF receptor and VEGF receptor kinase
activities. Although its ability to repress Mcl-1 has been
attributed to inactivating MAPK kinase and/or activating
GSK-3 [32], the exact mechanism remains unclear.
Nevertheless, this data suggests that Fbw7-deficient
T-ALL cell lines might require high levels of the Mcl-1
oncoprotein to evade apoptosis, a phenotype that has been
described previously as “oncogene addiction” [33]. Our
studies thus provide a basis for personalized medicine for
T-ALL patients as well as the rationale for developing
specific Mcl-1 antagonists, or agents that significantly
reduce Mcl-1 expression, to treat T-ALL patients.
the
cells Are resIstANt tO Abt-737
treAtmeNt
Fbw7-DeFIcIeNt t-All
The BH3 mimetic ABT-737 is a specific pan-inhibitor
of the Bcl-2 family of anti-apoptotic proteins, which is
reported to effectively kill leukemia cells, presumably
by disrupting the Bcl2/Bax complex and inducing the
Bak-dependent apoptotic pathway [34]. However,
leukemia cells with elevated Mcl-1 expression are found
to be refractory to ABT-737 treatment [35, 36] primarily
because ABT-737 fails to inactivate Mcl-1 due to a low
binding affinity [34]. Consistent with this, we found that
Fbw7-deficient T-ALL cells, which displayed a significant
increase in Mcl-1 expression, are much more resistant
than Fbw7-WT T-ALL cells in response to ABT-737
(Figure 4). We further showed that depletion of Mcl-1, or
reintroduction of Fbw7 into Fbw7-deficient T-ALL cells,
restored ABT-737 sensitivity. This suggests that increased
Mcl-1 expression is the determining factor that confers
Fbw7-deficient cells resistance to ABT-737. Although it
warrants further investigation, this work indicates that
Fbw7-deficient T-ALL patients may not respond well to
Figure 3: Fbw7-deficient T-ALL cells are “addicted” to high
levels of mcl-1 expression and are particularly sensitive to
the mcl-1 antagonist sorafenib.
A. In unstressed Fbw7-deficient T-ALL cells, induced expression of
the pro-survival factor Mcl-1 balances the pro-apoptotic effects of
elevated c-Jun, c-Myc and Notch-1 oncoproteins. However, the cells
become “addicted” to high expression levels of the Mcl-1 oncoprotein.
b. When Fbw7-deficient cells are treated with the Mcl-1 antagonist,
sorafenib, the pro-survival function of Mcl-1 is inhibited. On the other
hand, loss of Fbw7 leads to elevated expression of c-Jun, c-Myc and
Notch-1, which provokes cellular apoptosis. When the anti-apoptotic
effect of Mcl-1 is inhibited, cells undergo programmed cell death.
Therefore, compared to the WT-Fbw7 T-ALL cells, Fbw7-deficient
T-ALL cells are very sensitive to Mcl-1 antagonists.
Pro-apoptotic
Signal
Tumorigenesis
Cell Death
Cell Growth
Tumorigenesis
Cell Death
Pro-apoptotic
Signal
Cell Growth
c-Myc
c-Jun
Notch1
Mcl-1
c-Myc
c-Jun
Notch1
Mcl-1
A
Fbw7
Fbw7
B
Sorafenib

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ABT-737 treatment.
Fbw7 Is the physIOlOgIcAl e3
lIgAse thAt tArgets mcl-1 FOr
UbIqUItINAtION IN t-All
Besides Fbw7, other E3 ubiquitin ligases including
c-Mule [37] and β-TRCP [38] have also been implicated in
Mcl-1 stability control. However, we found that although
depletion of c-Mule leads to Mcl-1 upregulation in T-ALL,
this regulation is not contingent upon GSK3-dependent
phosphorylation of Mcl-1 [37, 38]. Most importantly,
unlike the frequent loss of Fbw7 found in T-ALL, no
correlation was found between the expression of c-Mule
and Mcl-1 in various T-ALL cell lines. These results
exclude a physiological role for c-Mule in regulating
Mcl-1 in T-ALL cells. Additionally, although ectopic
A
expression of β-TRCP promotes Mcl-1 destruction, no
β-TRCP-dependent induction of Mcl-1 ubiquitination
was observed. Furthermore, depletion of endogenous
Fbw7, but not endogenous β-TRCP, leads to a significant
induction of Mcl-1, rejecting the notion that β-TRCP
physiologically control Mcl-1 abundance in T-ALL cell
lines. Consistent with this finding, array CGH analysis
demonstrated a high frequency of Fbw7 loss [13], but not
simultaneous loss of β-TRCP1 and β-TRCP2 in the T-ALL
cell lines. These data together support the hypothesis that
the Fbw7 tumor suppressor, which is frequently lost in
T-ALL, is the physiological E3 ubiquitin ligase for Mcl-1.
However, it remains unclear whether in other tissue types
or cellular context, other than Fbw7, c-Mule or β-TRCP
will be the major force that governs Mcl-1 stability.
whether the pteN/pI3k/Akt AxIs Is
the UpstreAm sIgNAlINg pAthwAy
thAt gOverNs
UbIqUItINAtION OF mcl-1
Fbw7-meDIAteD
Our work indicates that loss of Fbw7 contributes to
Mcl-1 overexpression in T-ALL. However, only 20-30%
of T-ALL cases are estimated to possess inactive Fbw7
[13], thus it is critical to understand how Fbw7-mediated
Mcl-1 ubiquitination is physiologically regulated in vivo,
and how Mcl-1 expression is aberrantly elevated in WT-
Fbw7 genetic background, which accounts for 70-80%
of T-ALL cases. To this end, we found that in WT-Fbw7
genetic backgrounds, loss of the PTEN tumor suppressor,
which leads to inactivation of GSK3 kinase, also results in
elevated Mcl-1 expression in a similar fashion to Fbw7-
deficiency (data not shown). The PTEN phosphatase is
a negative regulator of the PI3K/Akt signaling pathway
and loss of PTEN is frequently observed in many types
of tumors including T-ALL [13, 39-41]. Since our recent
studies clearly demonstrated that GSK3 plays a critical role
for Fbw7-mediated Mcl-1 ubiquitination, we hypothesize
that any aberrant inactivation of GSK3, including loss of
PTEN activity [42], might phenocopy Fbw7 deficiency,
resulting in elevated Mcl-1 expression (Figure 5). Looking
forward, it is thus critical to determine, especially in the
setting of T-ALL, the molecular mechanisms by which
the PTEN/PI3K/Akt/GSK3 axis participates in Fbw7-
mediated destruction of Mcl-1, and to evaluate whether
Akt or mTOR inhibitors could be a novel anti-leukemia
therapeutic option to trigger apoptosis by decreasing Mcl-
1 protein abundance, especially for those who are PTEN-
deficient.
cONFlIct OF INterest
The authors have no conflict of interests to declare.
Figure 4: loss of Fbw7 leads to elevated mcl-1 expression,
which confers increased resistance to the pan-bcl-2 inhibitor
Abt-737.
A. In T-ALL cells with WT-Fbw7 genetic background, Mcl-1 stability
is governed by Fbw7 in a GSK3-dependent manner. Inhibition of the
Bcl-2 family of proteins with ABT-737 efficiently triggers apoptosis by
inducing cytochrome c release.
b. Due to structural differences, the pan-Bcl-2 inhibitor ABT-737
cannot efficiently inactivate Mcl-1 as it does to the rest of the Bcl-2
family of proteins. Therefore, loss of Fbw7 leads to elevated Mcl-1
expression, subsequently resulting in increased resistance to ABT-737.
Mitochondria
Mcl-1 Bcl2
cytochrome c
release
Apoptosis
ABT-737
Fbw7
Ub
Ub
Ub
Mitochondria
Mcl-1
Bcl2
cytochrome c
release
survival
ABT-737
Fbw7
B
26S
Proteasome
Degradation
Fbw7-WT
Fbw7-deficient

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AckNOwleDgemeNts
We thank Lixin Wan and Daming Gao for critical
reading of the manuscript, and the other members of
the Wei laboratory for useful discussions. W.W. is a
DOD Prostate Research Program New Investigator. Our
studies of Mcl-1 destruction were supported in part by
the Massachusetts Life Science Center New Investigator
award (W.W.), and by NIH grant GM089763 to W.W.
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