Download MUC1 oncoprotein is a druggable target in human prostate cancer

Published OnlineFirst November 3, 2009; DOI:10.1158/1535-7163.MCT-09-0646
3056
MUC1 oncoprotein is a druggable target in human prostate
cancer cells
Maya Datt Joshi,1 Rehan Ahmad,1 Li Yin,1
Deepak Raina,3 Hasan Rajabi,1 Glenn Bubley,2
Surender Kharbanda,3 and Donald Kufe1
1
Dana-Farber Cancer Institute; 2Beth Israel Deaconess Medical
Center, Harvard Medical School; and 3Genus Oncology, Boston,
Massachusetts
Abstract
Human prostate cancers are dependent on the androgen
receptor for their progression. The MUC1 heterodimeric
oncoprotein is aberrantly overexpressed in prostate cancers; however, it is not known if MUC1 is of functional
importance to these tumors. To assess dependence on
MUC1, we synthesized an inhibitor, designated GO-201,
which interacts directly with the MUC1-C subunit at its
oligomerization domain. Treatment of MUC1-positive
DU145 and PC3 prostate cancer cells with GO-201, and
not an altered version, resulted in inhibition of proliferation. GO-201 also induced necrotic cell death that was
associated with increases in reactive oxygen species, loss
of mitochondrial transmembrane potential, and depletion
of ATP. By contrast, GO-201 had no effect against
MUC1-negative LNCaP, CWR22Rv1, and MDA-PCa-2b
prostate cancer cells. Significantly, GO-201 treatment of
DU145 and PC3 xenografts growing in nude mice resulted
in complete tumor regression and prolonged lack of recurrence. These findings indicate that certain prostate cancer cells are dependent on MUC1-C for growth and
survival and that directly targeting MUC1-C results in
their death in vitro and in tumor models. [Mol Cancer Ther
2009;8(11):3056–65]
Introduction
The mucin 1 (MUC1) oncoprotein is aberrantly expressed at
high levels in human carcinomas (1) and has become an
Received 7/14/09; revised 8/14/09; accepted 9/8/09; published OnlineFirst
11/3/09.
Grant support: Dana-Farber/Harvard Cancer Center Nodal grant 2006-11NN, Department of Defense Prostate Cancer Idea Award W81XWH-08-10093, and National Cancer Institute grants CA97098 and CA42802.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Note: Supplementary material for this article is available at Molecular
Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute,
44 Binney Street, Dana 830, Boston, MA 02115. Phone: 617-632-3141;
Fax: 617-632-2934. E-mail: [email protected]
Copyright © 2009 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-09-0646
attractive target for the development of anticancer agents.
However, there have been no available small molecules to
date that directly target MUC1. In this regard, MUC1 is heterodimer that consists of NH 2 -terminal (MUC1-N) and
COOH-terminal (MUC1-C) subunits (2), and much of the
early work focused on MUC1-N, the mucin component.
Importantly, however, the transmembrane MUC1-C includes
a cytoplasmic domain that is sufficient for transformation (3,
4). Moreover, MUC1-C interacts with diverse effectors, such
as the epidermal growth factor receptor (5, 6), β-catenin (7),
p53 (8), IκB kinase β (9), and NF-κB p65 (10), which have been
linked to transformation. MUC1-C contains a CQC motif in
the cytoplasmic domain that is necessary for its oligomerization and thereby targeting of MUC1-C to the nucleus (11).
MUC1-C is also targeted to the mitochondrial outer membrane in a complex with heat shock protein 70/90 that is dependent on formation of MUC1-C oligomers (12–14).
Integration of MUC1-C in the mitochondrial outer membrane
blocks stress-induced loss of the mitochondrial transmembrane potential (ΔΨm; ref. 12). Consistent with this effect,
overexpression of MUC1 as found in human carcinomas
blocks the induction of apoptosis and necrosis in the cellular
response to DNA-damaging agents (12), reactive oxygen species (ROS; refs. 15, 16), hypoxia (17), and glucose deprivation
(18). Based on these observations, a direct inhibitor of MUC1C oligomerization was found to induce death of human breast
cancer cells growing in vitro and as tumor xenografts (19).
MUC1 is overexpressed in ∼60% of primary prostate cancers and 90% of lymph node metastases (20, 21). In addition,
86% of MUC1-positive primary prostate tumors were Gleason grade ≥7, supporting an association with more aggressive disease (20). Gene expression profiling of human
prostate cancers has also shown that MUC1 is highly expressed in subgroups with aggressive clinicopathologic features and an elevated risk of recurrence (22). Notably,
however, there are no reports that MUC1 contributes to
the malignant phenotype of prostate cancer cells. Indeed,
prostate cancer cells are dependent on androgen receptor
(AR) signaling for growth and survival (23). Moreover, progression of prostate cancer, despite treatment to abrogate
androgen action, occurs as a result of continued AR activation by mechanisms that include AR gene amplification and
mutations (23–25). Production of AR ligands by prostate
cancer cells (26), alterations in AR coactivators and repressors (27), and interactions with other signaling pathways
(28) have also been associated with progression of prostate
cancer to castrate-resistant disease. The importance of AR
signaling for growth of androgen-insensitive prostate cancer
cells has been further supported by the targeting of AR
function in in vitro and animal tumor models (29, 30). These
findings have provided the experimental basis for the development of new agents that inhibit AR function (31–33).
Mol Cancer Ther 2009;8(11). November 2009
Downloaded from mct.aacrjournals.org on March 28, 2012
Copyright © 2009 American Association for Cancer Research
Published OnlineFirst August 25, 2009; DOI:10.1158/0008-5472.CAN-09-0523
MUC1-C Oncoprotein Functions as a Direct Activator of the
Nuclear Factor- κB p65 Transcription Factor
Rehan Ahmad, Deepak Raina, Maya Datt Joshi, et al.
Cancer Res 2009;69:7013-7021. Published OnlineFirst August 25, 2009.
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Copyright © 2009 American Association for Cancer Research
Published OnlineFirst June 2, 2009; DOI:10.1158/0008-5472.CAN-09-0854
Research Article
Direct Targeting of the Mucin 1 Oncoprotein Blocks Survival
and Tumorigenicity of Human Breast Carcinoma Cells
Deepak Raina,1 Rehan Ahmad,1 Maya Datt Joshi,1 Li Yin,1 Zekui Wu,1 Takeshi Kawano,1
Baldev Vasir,1 David Avigan,2 Surender Kharbanda,1 and Donald Kufe1
1
Dana-Farber Cancer Institute and 2Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts
Abstract
The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed
by ∼90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of
breast cancer cells. We have synthesized a MUC1 inhibitor
(called GO-201) that binds to the MUC1 cytoplasmic domain
and blocks the formation of MUC1 oligomers in cells. GO-201,
and not an altered version, attenuates targeting of MUC1 to
the nucleus of human breast cancer cells, disrupts redox
balance, and activates the DNA damage response. GO-201 also
arrests growth and induces necrotic death. By contrast, the
MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor
xenografts was associated with loss of tumorigenicity and
extensive necrosis, which results in prolonged regression of
tumor growth. These findings show that targeting the MUC1
oncoprotein is effective in inducing death of human breast
cancer cells in vitro and in tumor models. [Cancer Res
2009;69(12):5133–41]
Introduction
Mucins are extensively O-glycosylated proteins that are predominantly expressed by epithelial cells. The secreted and membranebound mucins form a physical barrier that protects the apical
borders of epithelial cells from damage induced by toxins, microorganisms, and other forms of stress that occur at the interface
with the external environment. The transmembrane mucin 1
(MUC1) has no sequence similarity with other membrane-bound
mucins, except for the presence of a sea urchin sperm protein-enterokinase-agrin (SEA) domain (1). MUC1 is translated as a single
polypeptide and then undergoes autocleavage at the SEA domain
with the generation of two subunits that form a stable heterodimer
(2, 3). The MUC1 NH2 terminal subunit (MUC1-N) contains variable numbers of tandem repeats that are modified by O-glycosylation (4). MUC1-N extends beyond the glycocalyx of the cell and is
tethered to the cell surface through noncovalent binding to the
transmembrane MUC1 COOH terminal subunit (MUC1-C; ref. 5).
MUC1-C consists of a 58–amino acid extracellular domain, a 28–
amino acid transmembrane domain, and a 72–amino acid cyto-
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Current address for D. Raina, L. Yin, and S. Kharbanda: Genus Oncology, Boston,
MA 02118. Current address for T. Kawano: Jikei School of Medicine, Tokyo, Japan.
Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute, 44 Binney
Street, Dana 830, Boston, MA 02115. Phone: 617-632-3141; Fax: 617-632-2934; E-mail:
[email protected].
©2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-09-0854
www.aacrjournals.org
plasmic domain that interacts with diverse signaling molecules
(6). Shedding of MUC1-N into the protective physical barrier leaves
MUC1-C at the cell surface as a putative receptor to transduce intracellular signals that confer growth and survival (7, 8).
With transformation and loss of polarity, MUC1 is expressed at
high levels on the entire cell surface in a wide range of carcinomas
of the breast, lung, prostate, gastrointestinal tract, and other epithelia (9). Loss of restriction to the apical membrane allows for the formation of complexes with the epidermal growth factor receptor
(EGFR) and coactivation of EGFR-mediated signaling (7, 10). Overexpression of MUC1 by carcinoma cells is associated with the accumulation of MUC1-C in the cytosol and targeting of this subunit to
the nucleus (11–13) and mitochondria (14, 15). Importantly, the
MUC1 cytoplasmic domain (MUC1-CD) activates expression of
gene signatures that are predictive of both response to tamoxifen
and overall survival in breast cancer patients (16, 17). In this
context, oligomerization of MUC1-C is necessary for its nuclear targeting and interaction with diverse effectors (18). For example,
MUC1-CD functions as a substrate for c-Src (19), c-Abl (20), protein
kinase Cδ (21), and glycogen synthase kinase 3β (22) and interacts
directly with the Wnt pathway effector β-catenin (23, 24) and the
p53 tumor suppressor (25). Other work has shown that overexpression of the MUC1 heterodimer confers anchorage-independent
growth and tumorigenicity (12, 14, 25, 26), at least in part through
stabilization of β-catenin (24). Moreover, consistent with a survival
function for normal epithelial cells, overexpression of the MUC1
heterodimer confers resistance of carcinoma cells to stress-induced
apoptosis by a mechanism mediated in part through suppression of
intracellular reactive oxygen species (ROS; refs. 14, 27–29).
The present results show that targeting MUC1-C oligomerization blocks nuclear localization of MUC1-C and induces growth arrest and death of human breast cancer cells. The findings also
show that inhibiting MUC1-C is highly effective in the treatment
of human breast tumor xenografts in nude mice.
Materials and Methods
Cell culture. Human ZR-75-1 and MDA-MB-231 cell lines were grown in
RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (HIFBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Human MCF-7
breast cancer cells and 293 cells were grown in DMEM with 10% HI-FBS,
antibiotics, and 2 mmol/L L-glutamine. Primary human breast cancer cells
isolated from a malignant pleural effusion were cultured in RPMI 1640 containing 10% serum. Human MCF-10A breast epithelial cells were grown in
mammary epithelial cell growth medium (Lonza). Cells were treated with
GO-201, GO-202, or CP-1 peptides synthesized by MIT Biopolymer Laboratory and AnaSpec, Inc. Viability was determined by trypan blue exclusion.
Analysis of cell cycle distribution, apoptosis, and necrosis. Cells
were harvested, washed with PBS, fixed with 80% ethanol, and incubated
in PBS containing 40 μg/mL RNase and 40 μg/mL propidium iodide for
30 min at 37°C. Cell cycle distribution and sub-G1 DNA content were
5133
Cancer Res 2009; 69: (12). June 15, 2009
Downloaded from cancerres.aacrjournals.org on March 28, 2012
Copyright © 2009 American Association for Cancer Research
The Prostate 71:1299^1308 (2011)
Androgen Receptor Regulates Expression of the MUC1-C
Oncoprotein in Human Prostate Cancer Cells
Hasan Rajabi, Maya Datt Joshi, Caining Jin, Rehan Ahmad, and Donald Kufe*
Medical Oncology Department, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
BACKGROUND. The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in
human prostate cancers with more aggressive pathologic and clinical features. However,
the signals that regulate MUC1 expression in prostate cancer cells are not well understood.
METHODS. MUC1 expression was studied in androgen-dependent and -independent prostate cancer cell lines by quantitative RT-PCR, immunoblotting and assessment of MUC1
promoter activation. Chromatin immunoprecipitation (ChIP) studies were performed to assess
androgen receptor (AR) occupancy on the MUC1 promoter. Post-transcriptional regulation of
MUC1 expression was assessed by miR-125b-mediated effects on activity of the MUC1 30
untranslated region (30 UTR).
RESULTS. The present studies demonstrate that AR occupies a consensus AR element on the
MUC1 promoter in androgen-dependent LNCaP, but not in androgen-independent DU145 and
PC3, prostate cancer cells. The results further show that AR downregulates MUC1 gene transcription. Stable introduction of exogenous AR in PC3 (PC3/AR) cells and then silencing of AR
confirmed AR-mediated repression of the MUC1 promoter. AR signaling has also been shown
to drive miR-125b expression. The present studies further demonstrate that miR-125b suppresses MUC1 translation in LNCaP cells and that an anti-sense miR-125b upregulates expression of MUC1 protein. In addition, stable expression of miR-125b in DU145 cells resulted in
decreases in MUC1 levels.
CONCLUSIONS. These findings demonstrate that AR signaling regulates MUC1
expression by transcriptional and posttranscriptional mechanisms in prostate cancer cells.
Prostate 71: 1299–1308, 2011. # 2011 Wiley-Liss, Inc.
KEY WORDS:
prostate cancer; androgen-dependence; MUC1; androgen receptor; miR125b
INTRODUCTION
The mucin 1 (MUC1) heterodimer localizes to the
apical membrane of normal epithelial cells and is aberrantly overexpressed in diverse human carcinomas [1].
However, few insights are available regarding the
mechanisms responsible for the regulation of MUC1
expression in carcinoma cells [1]. MUC1 is translated as
a single polypeptide that is processed by autocleavage
into two subunits that in turn form a heterodimer. The
MUC1 N-terminal subunit (MUC1-N) contains highly
glycosylated tandem repeats that are characteristic of
mucin family members [1]. MUC1-N is positioned at
the cell surface in a complex with the MUC1 C-terminal
transmembrane subunit (MUC1-C) [1]. Release of
MUC1-N from the heterodimer leaves MUC1-C as a
potential transmembrane receptor for signaling stress
ß 2011Wiley-Liss,Inc.
Additional Supporting Information may be found in the online
version of this article.
Abbreviations: MUC1, mucin 1; MUC1-N, MUC1 N-terminal subunit; MUC1-C, MUC1 C-terminal subunit; AR, androgen receptor;
ARE, AR element; ChIP, chromatin immunoprecipitation; Luc,
luciferase.
Grant sponsor: Department of Defense Prostate Cancer Idea Award;
Grant number: W81XWH-08-1-0093.
D. Kufe holds equity in Genus Oncology and is a consultant to the
company.
*Correspondence to: Dr. Donald Kufe, Dana-Farber Cancer Institute,
44 Binney Street, Dana-830, Boston, MA 02115.
E-mail: [email protected]
Received 24 November 2010; Accepted 4 January 2011
DOI 10.1002/pros.21344
Published online 9 February 2011 in Wiley Online Library
(wileyonlinelibrary.com).
RESEARCH ARTICLE
CANCER
MUC1-C Oncoprotein Promotes STAT3 Activation
in an Autoinductive Regulatory Loop
Rehan Ahmad,* Hasan Rajabi,* Michio Kosugi, Maya Datt Joshi, Maroof Alam, Baldev Vasir,
Takeshi Kawano,† Surender Kharbanda,‡ Donald Kufe§
INTRODUCTION
Members of the signal transducer and activator of transcription (STAT)
family have been implicated in malignant transformation, as well as in
tumor cell survival, invasion, and metastasis (1). The STAT3 transcription
factor, which acts as an effector of the interleukin-6 (IL-6) inflammatory
response (2), is activated by Janus-activated kinase 1 (JAK1) phosphorylation of the IL-6 receptor and the subsequent recruitment and phosphorylation of STAT3 on a conserved tyrosine at position 705 (Tyr705) (1).
Phosphorylated STAT3 ( p-STAT3) undergoes dimerization and translocates to the nucleus to activate the transcription of target genes encoding
regulators of cell cycle progression (such as cyclin D1 and c-Myc) and
inhibitors of apoptosis (such as survivin and Bcl-xL) (3, 4). Constitutively
activated STAT3 induces transformation (5), and STAT3 activation has
been detected in various carcinomas and hematologic malignancies
(6–8), consistent with its involvement in the transcription of genes that
control cell proliferation and survival.
The unphosphorylated form of STAT3 has also been implicated in
transcriptional activation and linked to oncogenesis (9, 10). The gene encoding STAT3 is itself activated in response to IL-6–induced STAT3 phosphorylation (11), and the consequent increase in unphosphorylated STAT3
induces the transcription of additional genes, in part through STAT3
binding to the RelA subunit of the transcription factor nuclear factor
kB (NF-kB) (10). Small-molecule inhibitors of JAK1 and STAT3 have
anticancer activity in vitro and in animal models (12–16). Together, these
findings support a role for STAT3 signaling in tumorigenesis.
The mucin family of glycoproteins function in protecting the apical
surfaces of epithelial cells that interface with the external environment
and line ducts (17). The mucin 1 (MUC1) transmembrane oncoprotein
is overexpressed in various human carcinomas and certain hematologic
malignancies (17). Indeed, MUC1 overexpression is sufficient to induce
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115,
USA.
*These authors contributed equally to this work.
†Present address: Jikei University School of Medicine, Tokyo 105-8461, Japan.
‡Present address: Genus Oncology, Boston, MA 02118, USA.
§To whom correspondence should be addressed. E-mail: donald_kufe@dfci.
harvard.edu
transformation (18, 19). MUC1 is translated from a single transcript into
a polypeptide that undergoes autocleavage into two subunits, which in
turn form a heterodimer (17). MUC1 thus consists of an extracellular Nterminal mucin subunit (MUC1-N) that forms a complex with the transmembrane C-terminal subunit (MUC1-C) (20, 21). MUC1-C contains a
58–amino acid extracellular domain that interacts with galectin-3 and
thereby forms complexes with epidermal growth factor receptor (EGFR)
(22) and a cytoplasmic domain consisting of 72 amino acids including a
Cys-Glu-Cys (CQC) motif necessary for MUC1-C oligomerization and
function (23). The MUC1-C cytoplasmic domain (MUC1-CD) also
contains sites that function as substrates for phosphorylation by EGFR,
c-Met, c-Src, c-Abl, glycogen synthase kinase 3b, and protein kinase C
(17). MUC1-CD binds directly to the Wnt effector b-catenin and
contributes to activation of the Wnt pathway (17). c-Src phosphorylation
of MUC1-CD increases the binding of MUC1-CD to b-catenin (17).
MUC1-CD also interacts with the inhibitor of NF-kB kinase (IKK)
complex and RelA and contributes to activation of the NF-kB pathway
(24, 25). MUC1-C thus has the potential for multiple functions in cell
signaling and gene regulation as an adaptor or scaffold for interactions
with client proteins that, in certain settings, are regulated by MUC1-CD
phosphorylation (17).
With overexpression, MUC1-C accumulates in the cytoplasm and is
targeted to the nucleus by a mechanism involving importin-b and Nup62
(23, 26, 27) and to the mitochondria by a mechanism involving the
chaperones HSP70 (heat shock protein 70 kD) and HSP90 (17, 28, 29). In
the nucleus, MUC1-C interacts with various transcription factors, including
p53, and binds to the promoters of their target genes (17, 26, 27). As a
component of the mitochondrial outer membrane, MUC1-C attenuates loss
of the transmembrane potential in response to genotoxic stress (17, 28).
Consistent with these findings, MUC1-C confers resistance to death in
the cellular response to DNA damage, reactive oxygen species, hypoxia,
or activation of the death receptor superfamily (17). Thus, like STAT3,
MUC1-C has been linked to pathways that lead to malignant transformation
and attenuation of cell death.
Here, we show that the MUC1-CD binds directly to JAK1 and STAT3
and that MUC1-C promotes JAK1-mediated phosphorylation of STAT3.
Activated STAT3 induces expression of the MUC1 gene, and, in an auto-
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Signal transducer and activator of transcription 3 (STAT3) is activated in human breast cancer and
other malignancies. Mucin 1 (MUC1) is a heterodimeric cell surface glycoprotein that is overexpressed
in human carcinomas and, like STAT3, promotes cell survival and induces transformation. We found
that in breast cancer cells, the MUC1 carboxyl-terminal receptor subunit (MUC1-C) associates with the
gp130–Janus-activated kinase 1 (JAK1)–STAT3 complex. The MUC1-C cytoplasmic domain interacted
directly with JAK1 and STAT3, and MUC1-C was necessary for JAK1-mediated STAT3 activation. In turn,
MUC1-C and activated STAT3 occupied the promoter of MUC1, and MUC1-C contributed to STAT3mediated activation of MUC1 transcription. The MUC1-C inhibitor GO-201 blocked the MUC1-C interaction
with STAT3, thereby decreasing MUC1-C and STAT3 occupancy on the MUC1 and STAT3 promoters and
activation of STAT3 target genes, including MUC1 itself. These findings indicate that MUC1-C promotes
STAT3 activation and that MUC1-C and STAT3 function in an autoinductive loop that may play a role in
cancer cell survival.