Lehman NL, Tibshirani R, Hsu JY, Natkunam Y, Harris BT, West RB et al.. Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors. Am J Pathol 170: 1793-1805

Article (PDF Available)inAmerican Journal Of Pathology 170(5):1793-805 · June 2007with50 Reads
DOI: 10.2353/ajpath.2007.060767 · Source: PubMed
Abstract
The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.
Tumorigenesis and Neoplastic Progression
Oncogenic Regulators and Substrates of the
Anaphase Promoting Complex/Cyclosome Are
Frequently Overexpressed in Malignant Tumors
Norman L. Lehman,*
Rob Tibshirani,
Jerry Y. Hsu,
§
Yasodha Natkunam,*
Brent T. Harris,*
Robert B. West,*
Marilyn A. Masek,* Kelli Montgomery,*
Matt van de Rijn,* and Peter K. Jackson*
§
From the Departments of Pathology,* Health Research and Policy,
and Statistics,
the Division of Neuropathology,
and the Program
in Cancer Biology,
§
Stanford University, Stanford, California
The fidelity of cell division is dependent on the accu-
mulation and ordered destruction of critical protein
regulators. By triggering the appropriately timed,
ubiquitin-dependent proteolysis of the mitotic regu-
latory proteins securin, cyclin B, aurora A kinase,
and polo-like kinase 1, the anaphase promoting com-
plex/cyclosome (APC/C) ubiquitin ligase plays an es-
sential role in maintaining genomic stability. Misex-
pression of these APC/C substrates, individually, has
been implicated in genomic instability and cancer.
However, no comprehensive survey of the extent of
their misregulation in tumors has been performed.
Here, we analyzed more than 1600 benign and malig-
nant tumors by immunohistochemical staining of tis-
sue microarrays and found frequent overexpression
of securin, polo-like kinase 1, aurora A , and Skp2 in
malignant tumors. Positive and negative APC/C regu-
lators, Cdh1 and Emi1, respectively, were also more
strongly expressed in malignant versus benign tu-
mors. Clustering and statistical analysis supports the
finding that malignant tumors generally show broad
misregulation of mitotic APC/C substrates not seen in
benign tumors, suggesting that a “mitotic profile” in
tumors may result from misregulation of the APC/C
destruction pathway. This profile of misregulated mi-
totic APC/C substrates and regulators in malignant tu-
mors suggests that analysis of this pathway may be
diagnostically useful and represent a potentially impor-
tant therapeutic target.
(Am J Pathol 2007, 170:1793–1805;
DOI: 10.2353/ajpath.2007.060767)
Tumor progression is characterized by misregulation of
critical growth regulatory mechanisms. Typically, activa-
tion of growth factor pathways, eg, through tyrosine ki-
nases or growth factors up-regulating cyclin D, and loss
of growth regulatory tumor suppressors, eg, pRb, p16,
and p53, directs unscheduled cell division.
1
In many
tumors, neoplastic transformation is strongly linked to the
development of chromosome instability, leading to acti-
vation of the aforementioned and additional oncogenic
processes.
Recent studies have demonstrated that failure of nor-
mal chromosome segregation leading to subsequent mi-
totic catastrophe is a central mechanism among events
leading to chromosome or genomic instability. Mitotic
catastrophe is often linked to a failure of cytokinesis,
giving rise to tetraploid or aneuploid cells. Tetraploidy is
thought to provide a buffer against genetic loss in
genomically unstable cells, having recently been shown
to be the preferred pathway for cells that fail mitosis
2
and
to otherwise independently lead to a tumorigenic state in
p53-null cells.
3
Mitotic catastrophe also leads to aneu-
ploidy, possibly through tetraploid intermediates, and the
genomic rearrangement typically seen in malignant
tumors.
Misregulation of specific mitotic regulators can drive
mitotic catastrophe in model genetic organisms, in cul-
tured mammalian cells, and in mouse models. Notably,
over- or underexpression of the mitotic kinases aurora A
and polo-like kinase 1 (Plk1) and the chromosome seg-
regation regulator securin can each give rise to mitotic
catastrophe.
4–7
Each of these proteins, along with the
mitotic entry regulator Skp2,
8,9
have been suggested to
Supported by National Institute of Neurological Disorders and Stroke
grant K08 NS45077 (to N.L.L.) and National Institute of General Medical
Sciences grant RO1 GM60439 (to P.K.J.).
Accepted for publication February 13, 2007.
Supplemental material for this article can be found on http://ajp.
amjpathol.org.
Current address of P.K.J.: Genentech, Inc., South San Francisco, CA.
Address reprint requests to Norman L. Lehman, Department of Pathol-
ogy, MC5324, Stanford University Medical Center, 300 Pasteur Dr., Stan-
ford, CA 94305. E-mail: nlehman@stanford.edu.
The American Journal of Pathology, Vol. 170, No. 5, May 2007
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.060767
1793
be oncogenic, possibly by driving chromosomal rear-
rangement. Of interest, these proteins are substrates of
the anaphase promoting complex or cyclosome (APC/C),
the E3 ubiquitin ligase controlling destruction of mitotic
cyclins, and other mitotic regulators, among other pro-
teins.
10
A biologically consistent model is that the care
-
fully timed destruction of these proteins in mitosis reflects
the importance of restricting their abundance and that
their overexpression disrupts the timing of mitotic events.
The APC/C is a multisubunit ubiquitin ligase that rec-
ognizes critical RXXL or KEN amino acid motifs (degrons)
within protein substrates to assemble polyubiquitin
chains on these substrates, thereby targeting them to the
26S proteasome for proteolytic destruction. The APC/C
exists in two forms depending on its associated activator
protein, Cdc20 or Cdh1 (homologous to the Drosophila
protein Fizzy-related and should not be confused with
cadherin E, previously referred to as Cdh1 for cadherin
1). The APC/C
Cdc20
functions in early mitosis to destroy
cyclin A and securin and is regulated by the mitotic
spindle assembly checkpoint (discussed below). The
APC/C
Cdh1
functions later in mitosis to direct the destruc
-
tion of a host of mitotic regulators, thereby promoting
mitotic exit.
To achieve the critical timing of substrate destruc-
tion, the APC/C itself must be tightly regulated. At the
G
1
/S transition, the APC/C
Cdh1
ligase is inhibited by the
zinc-binding protein Emi1
11,12
(Figure 1
). This allows
APC/C substrate proteins important for progression of
S phase and early mitosis to accumulate.
13,14
In early
mitosis, Emi1 is phosphorylated by Plk1,
15
which trig
-
gers its ubiquitination by the SCF
TrCP
E3 ubiquitin
ligase.
14
This in turn causes the activation of the APC/C
in early prometaphase and cell cycle progression
through early mitosis.
During late prometaphase and metaphase, a group of
proteins comprising the mitotic spindle checkpoint inhib-
its APC/C
Cdc20
activity. The function of the spindle check
-
point is to prevent chromosome segregation from occur-
ring before the metaphase mitotic spindle has perfectly
formed, to ensure the equal segregation of sister chro-
matids to each daughter cell.
16
The APC/C activator
Cdh1 is itself an APC/C substrate,
17
further exemplifying
the tight and complex regulation of the APC/C. For mitotic
progression to occur smoothly, the APC/C initiates the
sequential, timed destruction of cyclin A, securin, cyclin
B, aurora A, aurora B, Plk1, and Cdh1 (Figure 1). The
precise details of how these specific events are orga-
nized are currently the subject of intense study.
Given the exquisite timing of events that is necessary
during mitosis, it is not surprising that misregulation of the
APC/C appears linked to catastrophic events in mitosis.
Illustrating this theme, misregulation of either of two crit-
ical regulators of the APC/C, the Mad2 spindle check-
point protein and the APC/C inhibitor Emi1, induces mi-
totic catastrophe.
14,18
Because inhibition of the APC/C
can stabilize a host of key mitotic regulators, including
aurora A, securin, Plk1, and cyclins, even subtle misregu-
lation of the APC/C has the potential to cause genomic
instability. The APC/C substrate Skp2 is also an important
cell cycle control protein. Skp2 seems to regulate both
the G
1
/S and G
2
/M transitions, where, as a subunit of an
SCF E3 ligase, it is required for ubiquitination of the
cyclin-dependent kinase inhibitor p27.
19
Recent studies suggest that APC/C regulation and the
control of cyclin accumulation may be linked to growth
factor pathways frequently misregulated in cancer. Nota-
bly, both the Emi1 and Mad2 APC/C inhibitors have been
shown to be targets of E2F transcription factors,
13,20
potentially linking the frequent misregulation of the cyclin
D/retinoblastoma/E2F pathway to APC/C misregulation.
We previously found that Emi1 mRNA was up-regulated
in several human tumors,
13
which led us to suspect that
APC/C misregulation might be a common event in can-
cer. We have also shown that Emi1 overexpression leads
to unscheduled cell proliferation, tetraploidy, and chro-
mosomal instability in p53-deficient cells.
21
In p53 wild-
type cells, the induction of tetraploidy and aneuploidy by
overexpression of APC/C inhibitors like Emi1 typically
leads to G
1
arrest or apoptosis. In p53 checkpoint-defi
-
cient cells, the continuation of unchecked proliferation in
the face of severe chromosome rearrangement by mitotic
catastrophe probably results in the striking aneuploidy
seen in many malignant tumors.
Here, we examined the extent of APC/C pathway
misregulation in human neoplasms by a broad survey
of the protein expression of APC/C substrates and the
APC/C regulators Emi1 and Cdh1 in all major types of
human tumors using immunohistochemical analysis of
tissue microarrays (TMAs).
22
Analysis of more than
1600 benign and malignant tumors revealed over-ac-
cumulation of securin, Plk1, aurora A, Cdh1, and Emi1
in malignant tumors but generally not in benign tumors.
Strikingly, the misregulation of these mitotic regulators
was strongly linked in specific classes of highly malig-
nant tumors. We propose that misregulation of the
mitotic destruction pathway leads to a “mitotic APC/C
substrate profile” of misregulation in malignant tumors
and that this profile may be of predictive value in
diagnosis and therapeutic response.
Growth
Signals
pRb
APC/C
Mitotic
Catastrophe
p53
Emi1
E2F
S phase
Normal
Growth
Abnormal
Growth
Cyclin A
Skp2
Aurora A
Plk1
Cyclin B
E2F
Cyclin E
Cyclin D
Mitosis
Normal
Mitosis
M
i
sregula
tionM
i
sregula
tion
Securin
Figure 1. Model for pRb- and APC/C-dependent control of S phase and early
mitosis. G
1
proliferation control genes upstream of Emi1 (shown in blue)
regulate the E2F-dependent expression of Emi1 and certain APC/C substrates
(cyclin A, Plk1, and securin). Accumulation of Emi1 stabilizes APC/C sub-
strates vital for progression through S phase (blue ) and mitosis (pink). When
overexpressed Emi1, or the mitotic control APC/C substrates Skp2, Plk1,
securin, or aurora A can induce mitotic catastrophe. In the absence of p53,
these genomically unstable cells survive and lead to tumor formation.
1794 Lehman et al
AJP May 2007, Vol. 170, No. 5
Materials and Methods
Short Interferring RNA (siRNA)
siRNA for human Emi1 target sequence 5-AAACU-
UGCUGCCAGUUCUUCA-3 and control siRNA for green
fluorescent protein target sequence 5-GGCTACGTC-
CAGGAGCGCACC-3 (Dharmacon RNA Technologies,
Lafayette, CO) were transfected into the HCT116 cells
shown in Figure 2 using Oligofectamine (Invitrogen,
Carlsbad, CA) in serum-free culture media for 4 hours.
Immunohistochemistry
Immunohistochemical staining was performed on 4-
m
paraffin-embedded tissue sections and TMA slides. An-
tigen retrieval was by citrate (pH 6.0), ethylenediamine
tetraacetic acid (pH 8.0), or Tris (pH 10.0) buffer and
microwave heating. Endogenous peroxidase and non-
specific binding (when necessary) were blocked using
3% hydrogen peroxide and Power Block (Biogenix, San
Ramon, CA), respectively. The secondary antibody was
Envision Plus (Dako, Glostrup, Denmark) anti-mouse or
anti-rabbit-horseradish peroxidase. 3,3-Diaminobenzi-
dine was the chromagen. The counterstain was Mayer’s
hematoxylin. HCT116 cells in Figure 2 were fixed in ace-
tone; 3-amino-9-ethyl carbazole was the chromagen, and
Gills’s hematoxylin was the counterstain. Primary anti-
bodies were as follows: affinity-purified rabbit anti-human
Emi1 as previously described
13
; anti-human aurora-A
monoclonal antibody
23
; anti-human cyclin E monoclonal
antibody (Novacostra, Newcastle, UK); rabbit anti-human
cyclin A (H-432), cyclin B1 (H-433), cyclin D1 (H-295),
cyclin D2 (C-17), cyclin E (C-19) (used for the breast TMA
only), and E2F-3 (C-18) (Santa Cruz Biotechnologies,
Santa Cruz, CA); rabbit anti-human phospho-pRb (Ser
807/811) (Cell Signaling Technologies, Danvers, MA);
anti-human
-catenin, p27
KIP1
and Skp2 monoclonal an
-
tibodies, rabbit anti-human securin (PTTG1), anti-Plk1,
and anti-Cdh1 antibodies (Zymed Laboratories, South
San Francisco, CA); and anti-Ki67, anti-Bcl2, anti-HER2,
and anti-ER
mouse monoclonal antibodies (Dako).
Staining parameters for these antibodies are summarized
in Table 1.
Tissue Microarrays
TMAs composed of 0.6-mm cores were constructed from
formalin-fixed and paraffin-embedded human tissues
(with the exception that the tissue was first fixed with
ethanol for the breast TMA), immunostained, and scored
by a pathologist as previously described.
24
TMAs were
scored according to the number of tumor cells demon-
AB
Figure 2. Validation of anti-Emi1 immunohistochemical staining. A: An
Emi1-immunopositive ovarian clear cell carcinoma was stained with anti-
Emi1 antibody showing characteristic cytoplasmic Emi1 immunoreactivity
(top) or antibody preincubated with recombinant antigen eliminating Emi1-
specific immunostaining (bottom). B: HCT116 cells transfected with control
siRNAs specific for green fluorescent protein (top) or siRNAs specific for
Emi1 (bottom) were fixed and immunostained for Emi1. Cells transfected
with Emi1 siRNA express considerably less Emi1 as indicated by the decrease
in red AEC chromagen labeling of the cells and elimination of the Emi1 band
by Western blot (right).
Table 1. Immunostaining Parameters for Primary Antibodies Used
Target Species and type Pretreatment and dilution Staining pattern
Aurora A Mouse monoclonal Citrate 1:25 Cytoplasmic
Bcl2 Mouse monoclonal Citrate 1:50 Nuclear
-Catenin Mouse monoclonal Citrate 1:25 Nuclear and cytoplasmic
Cdh1 Rabbit polyclonal Citrate 1:20 Cytoplasmic
Cyclin A Rabbit polyclonal Citrate 1:100 Nuclear
Cyclin B1 Rabbit polyclonal EDTA 1:100 Nuclear
Cyclin D1 Rabbit polyclonal Citrate 1:50 Nuclear and cytoplasmic
Cyclin D2 Rabbit polyclonal Citrate 1:25 Nuclear and cytoplasmic
Cyclin E Rabbit polyclonal Citrate 1:250 Nuclear
Cyclin E Mouse monoclonal Citrate 1:30 Nuclear
E2F-3 Rabbit polyclonal Citrate 1:400 Nuclear
Emi1 Rabbit polyclonal Citrate 1:1000 Cytoplasmic
ER
Mouse monoclonal Citrate* Nuclear
HER-2 Mouse monoclonal Citrate 1:600 Entire membrane
Ki-67 Mouse monoclonal Citrate 1:100 Nuclear
c-Myc Mouse monoclonal Citrate 1:200 Nuclear
Phos-pRb Rabbit polyclonal Citrate 1:50 Nuclear
Plk1 Rabbit polyclonal Tris 1:20 Cytoplasmic
p27 Mouse monoclonal Tris 1:1000 Nuclear
Securin Rabbit polyclonal Citrate 1:50 Cytoplasmic
Skp2 Mouse monoclonal Tris 1:500 Nuclear
EDTA, ethylenediamine tetraacetic acid.
*Prediluted manufacturer’s kit.
APC/C Substrates Are Overexpressed in Tumors 1795
AJP May 2007, Vol. 170, No. 5
strating specific immunoreactivity for a given primary an-
tibody within a given sample core. Immunostaining was
defined as negative (3% of tumor cells positive), weak
(3 to 29% of tumor cells positive), and strong (30%
tumor cells positive). Each tumor or tissue sample was
represented once on the TMAs, except for the connective
tissue tumor TMA where each sample was represented
by two cores. In the latter case, the greatest percentage
of positive cells of either core was scored. Data were
processed, and complete-linkage hierarchical clustering
was performed using samples in which 80 to 100% of
marker data were available using Cluster and Tree View
software.
24
To facilitate comparison of Emi1 and APC/C
substrate accumulation with that of other markers, we
weighted clustering on Emi1 expression, which ordered
the tumors into Emi1-negative and -positive groups. This
yielded identical cluster dendrograms of tumor markers
compared with unweighted clustering. The neural TMA
contained 180 tumors; the lymphoma TMA, 265 prolifer-
ative lesions; the breast TMA, 255 proliferative lesions;
two cancer TMAs, 523 tumors of diverse tissues; and the
connective tissue TMA, 460 tumors. TMAs contained vari-
able numbers of control and normal tissue cores.
Statistical Analysis
To assess significance, we tabulated Pearson corre-
lation coefficients and corresponding two-sided P
values, based on normal theory,
25
for immunoposi-
tivity for protein markers relative to each other
within TMAs (Supplemental Table 1, see http://ajp.
amjpathol.org).
Results and Discussion
The APC/C Regulator Emi1 Is Highly Expressed
in Tumors
To determine the possible extent of APC/C misregulation
in human cancer, we used immunohistochemical staining
to examine whether the APC/C regulators Emi1 and Cdh1
and several APC/C substrates were overexpressed in
tumors. We began by extending our initial observation of
Emi1 mRNA up-regulation in human tumors to the protein
level. Validation of our anti-human Emi1 antibodies is
shown in Figure 2.
Lymphoma (DLBCL)
T-Cell Lymphoma
Normal Lymph Node
Me
n
ingioma
C
h
o
r
do
ma
Oligodendroglio
m
a
Endome
t
rial Can
cer
Retinoblast
om
a
Lung Adenocarcinoma
Renal Cell Cancer
Ductal Cancer In Situ
Breast Fibroadenoma
Hepatic Carcinoma
Ewing's Sarcoma
Normal Breast
Seminom
a
Ep
e
ndym
om
a
Retinoblastoma
Ovary Clear Cell Ca
Ductal Cancer
Melanoma
Figure 3. Analysis of Emi1 protein expression in human tumors. Emi1 is highly expressed in retinoblastoma ( eye whole mount and retinoblastoma rosette) and
other major tumor types (TMA cores). The viable peripheral tumor tissue within the eye whole mount is Emi1 immunopositive, whereas the necrotic centeris
nonreactive. Normal lymph nodes and breast tissue exhibit less intense Emi1 staining than lymphomas and breast ductal cancer, respectively. Benign fibrocystic
breast tissue and endometrial stroma show very little Emi1 staining in the ductal carcinoma in situ and endometrial cancer cores, respectively. Positive
immunoreactivity is indicated by the brown diaminobenzidine chromagen.
1796 Lehman et al
AJP May 2007, Vol. 170, No. 5
Since Emi1 transcription is driven by E2F, we expected
that Emi1 would be highly expressed in retinoblastomas,
where E2Fs are not inhibited by pRb, and found strong
Emi1 immunopositivity in retinoblastomas (Figure 3).
Because overexpression of Emi1 protein in tumors
would be expected to lead to inappropriate APC/C inhi-
bition and hyperaccumulation of APC/C substrates, we
next used TMAs
22
to screen a large sample of human
tumors for Emi1 and APC/C substrate protein accumula-
tion. Immunohistochemical staining of TMAs has several
advantages over RNA-based methods for analyzing
gene expression in tumors, particularly when examining
components and substrates of the ubiquitin proteasome
system.
26
Most importantly, relative message levels often
do not accurately reflect relative protein levels in tumors,
and TMAs allow semiquantitative measurement of protein
levels within tumor cells specifically, without a confound-
ing contribution from nontumorous stromal cells, as is the
case with other methods.
26
Background staining can be
a limitation of immunohistochemistry, but this can gener-
ally be minimized by careful optimization of antibody
dilutions and antigen retrieval techniques and by inter-
pretation performed by experienced pathologists using
consistent criteria for immunopositivity between various
tissues for a given antibody.
We found that Emi1 protein is highly expressed in a
significant fraction of human neoplasms. Examples of
TMA immunostaining for Emi1 and other markers are
shown in Figure 3 and Supplemental Figures 1 and 2, see
http://ajp.amjpathol.org. A summary of the immunohisto-
chemical protein expression of Emi1, Cdh1, and the on-
cogenic APC/C substrates securin, Plk1, Skp2, and au-
rora A in many of the more common human tumors is
presented in Figure 4. Here, tumors are grouped accord-
ing to a classification system based on common devel-
opmental origin.
27
The figure depicts the percentage of
immunopositive individual tumor specimens for each pro-
tein marker.
Notably, we found that 92% of renal cell carcinomas,
80% of cervical adenocarcinomas, 79% of hepatocellular
carcinomas, 68% of oligodendrogliomas, 64% of lung
adenocarcinomas, 62% of endometrial cancers, 55% of
melanomas, and many lymphomas are Emi1 immunopo-
sitive. All germ cell tumors and all clear carcinomas of the
ovary examined strongly expressed Emi1. A large frac-
tion of other carcinomas, nonastrocytic neural tumors,
and some sarcomas were also Emi1 immunopositive.
Many astrocytomas, gastrointestinal adenocarcinomas,
sarcomas, and most low-grade connective tissue tumors
were Emi1 negative (Figure 4).
A number of trends were observed in specific classes
of tumors. First, among neural and connective tissue
neoplasms, benign tumors (indicated in blue in Figure 4)
were typically Emi1-negative, whereas a subset of malig-
nant tumors were Emi1-positive.
Second, in lymphomas, Emi1 expression generally
paralleled increasing tumor grade. Here, 59% of World
Health Organization grade I follicular lymphomas were
Emi1 immunopositive compared with 82% of grade III
follicular lymphomas and 81% of diffuse large B-cell lym-
phomas. Peripheral T-cell lymphomas, a particularly ag-
gressive lymphoma, were 100% Emi1 immunopositive.
Third, in some cancers, notably colon and breast can-
cer, substantial numbers of tumors lacked Emi1 immuno-
reactivity. Although some Emi1 immunoreactivity was
present in corresponding normal tissues, it was of lower
intensity and of a more compartmentalized manner within
individual cells compared with staining in Emi1-positive
tumors (Figure 3; Supplemental Figures 1 and 2, see
http://ajp.amjpathol.org). The corresponding nonmalig-
nant neoplasms, breast ductal papilloma and fibroade-
noma, and premalignant neoplasm, colon tubular ade-
noma, showed Emi1 staining of intermediate intensity.
In breast cancer, more Emi1-positive tumors were low
grade (43.8% grade 1, 31.3% grade 2, and 25.0% grade
3), whereas more Emi1-negative tumors tended to be
higher grade (15.8% grade 1, 47.4% grade 2, and 36.8%
grade 3). Emi1 (Fbx05) maps to chromosomal region
6q25,
28
close to the estrogen receptor
and Parkin
genes, which frequently undergo loss of heterozygosity in
breast cancers.
29
Likewise, papillary serous ovarian car
-
cinomas, in which chromosomal region 6q25 is also fre-
quently deleted, were Emi1 negative in approximately
50% of cases (Figure 4; Supplemental Figure 3, http://
ajp.amjpathol.org). In these tumors, Emi1 is likely impor-
tant at some early phase of tumor progression, but later
Emi1 loss may provide a second step in tumor progres-
sion or may simply be a consequence of further genomic
instability. Of biological note, the magnitude of differ-
ences in Emi1 protein levels between low- and high-
Emi1-expressing ovarian tumors determined by Western
blot (Supplemental Figure 3, http://ajp.amjpathol.org) was
greater than the level of Emi1 overexpression that re-
sulted in chromosomal instability in cell culture models.
21
APC/C Substrates Are Frequently
Overexpressed in Malignant Tumors
Because the extent of misregulation of APC/C substrates
in human neoplasms is largely unknown, we surveyed
APC/C misregulation in cancers by immunostaining
TMAs for the APC/C substrates cyclin A, cyclin B, se-
curin, aurora A, Plk1, Skp2, and Cdh1. To look for corre-
lations, we analyzed these data using complete-linkage
hierarchical clustering adapted for TMAs.
24
In many tu
-
mors with elevated Emi1 protein, several or all of the
APC/C substrates clustered together, consistent with the
model that Emi1 causes their stabilization. These include
the following tumors: lung and cervical adenocarcino-
mas; lung, esophageal and head and neck squamous
cell cancers; melanomas; lymphomas; urothelial transi-
tional cell tumors; seminomas; ovarian clear cell carcino-
mas; several malignant neural tumors; and some sarco-
mas (Figures 4– 6; Supplemental Tables 1 and 2, see
http://ajp.amjpathol.org). Thus, it seems that APC/C mis-
regulation by Emi1 overexpression or other factors may
direct a broad program of APC/C substrate stabilization
in tumors.
Specific APC/C substrates, notably Skp2, fail to follow
the pattern in some tumors (Skp2 staining is not prevalent
APC/C Substrates Are Overexpressed in Tumors 1797
AJP May 2007, Vol. 170, No. 5
Lymphoma
28
55
55
72
46
80
64
63
76
72
80
81
20
0
0
40
59
81
60
34 79 84
77
82
Follicular, Grade III
Follicular, Grade II
Follicular, Grade I
Diffuse Large B-Cell
Splenic Marginal Zone
75
88
100
35
75
93
25
24
80
62
50
39
Endometrial Cancer
Cervical Adenocarcinoma
33 67
83
100
100
60
Ovarian Clear Cell Ca.
91
20
100
82
100
27
Seminoma
Adrenocortical Cancer
50
75
50
100 100
100
80
100
100
50
0
36 32 74
67
44 23
Transitional Cell Cancer
Cervical Squamous Ca.
76
96 85
7152
52
Ovarian Papillary Cancer
7
73
62
92
0
Renal Cell Carcinoma
7
0
71
57
3850
100
70
100
70
CLL/SLL
Lymphoblastic T-Cell
Peripheral T-Cell
100
60
100
100
86
100
100
Germ Cell
Connective Tissue
19
0
60
17
8
41
0
25
0
41
7
39
36
25
34
50
86
64
16
23
33
97
67
94
69
92
79
33
73
100
100
71
57
44
25
Rhabdomyosarcoma
Leiomyosarcoma
Osteosarcoma
Malig. Fibrohistiocytoma
GI Stromal Tumor
Synovial Sarcoma
Myxofibrosarcoma
0 23 32
52
77
Solitary Fibrous Tumor
100
63
38
63
100
Ewing’s Tumor
Medulloblastoma
PNET/Neuroblastoma
29 25 75 50
75
0
0
100
0
100
33
33
33
67
0
Nephroblastoma
EMBRYONIC
Leiomyoma
Granular Cell Tumor
DFSP
10
11
11
88
67
0
0
0
100
0
0
0
0
100
0
Liposarcoma
Atypical Lipoma
0
0
33
0
20
38
30 83 83
63
MESODERM
Sub-Coelomic
Coelomic
Primitive Differentiating
Hepatic Adenocarcinoma
Esoph
.
Adenocarcinoma
Esoph
.
Squamous Ca.
Pancreas Adenoca
Gastric Adenocarcinoma
75
63
8
25
29
62
7
50
13
43
21
25
30
20
23
92
86
75
83
76
82
43
57
75
32
50
Colon Adenoca. Grade I
50
25
14
46
79
44 75
92
38
10
17
86 83
91
8
28
79
87
70
29
83
100
71 1717
Breast Lobular Cancer
Breast Ductal
Cancer
Breast CIS
Breast Fibroadenoma
Thyroid Papillary Ca.
12
0
47
100
27
ENDODERM/ECTODERM
Neural Crest
0
70
0
0
89
0
0
67
0
0
10
18
0
38
0
Meningioma, Grade I
MPNST
Neurofibroma
Schwannoma
0
88
17
86
89
43
43
86
Oligodendroglioma
Ependymoma
100
68
80
45
90
95
76
20
0
29
43
0
44
40
0
0
0
25
40
40
0
40
Astrocytoma, Grade III
Astrocytoma Grade II
Astrocytoma Grade I
40
25
35
55
50
76 71
67
Glioblastoma,Grade IV
Neural Tube
Melanoma
Lung Squamous Cancer
Head/Neck Squamous
77
74
69
65
58
92
64
83
67
38
26
Lung Adenocarcinoma
71
18
90
57 25
64
89 62
71
64
55
43
29 14 79
93 50
100
Meningioma, Grade II-III
100
10
13
100
100
88
Parenchymal Epithelium
Surface Epithelium
NEUROECTODERM
53
15
20
88
67
19 3
Prostate Cancer
Endocrine
50
29
25
60 80
20
0
20
39
86
24
67
50 54
27 82
25
100
71
64
25
Colon Adenoca. Grade II
Colon Adenoca. Grade III
Figure 4. Summary of TMA analysis of Emi1 and APC/C substrate protein expression in human tumors. TMAs representing different classes of tumors, grouped
according to developmental tissue origin, were analyzed by immunohistochemical staining. Numbers in boxes indicate the percentage of positive tumors (specific
numbers of immunopositive and total tumors surveyed are summarized in Supplemental Table 2, see http://ajp.amjpathol.org). Green indicates a low percentage
of immunopositive tumors (33% of cases); dark red, an intermediate percentage (33 to 66%); and bright red, a high percentage of Emi1-positive tumors (66% ).
Benign tumors are highlighted by the blue boxes and less aggressive malignant tumors by yellow boxes. World Health Organization grades are indicated for
astrocytomas and follicular lymphomas. Histopathological grades are also listed for colon adenocarcinomas. Adenoca, adenocarcinoma; Ca, carcinoma; CIS,
carcinoma in situ; DFSP, dermatofibrosarcoma protuberans; esoph., esophageal; GI, gastrointestinal; HN, head and neck; MPNST, malignant peripheral nerve
sheath tumor; PNET, primitive neuroectodermal tumor.
1798 Lehman et al
AJP May 2007, Vol. 170, No. 5
in hepatic, pancreatic, or renal carcinomas, despite
strong involvement of other APC/C substrates), possibly
because of genomic rearrangement or regulatory differ-
ences, eg, tissue-specific signaling factors or repressors.
This is supported by the fact that Skp2 was infrequently
overexpressed in all of the different types of breast tu-
mors and all of the tumors derived from organ parenchy-
mal epithelium (Figure 4).
APC/C misregulation by Emi1 is closely linked to the
pRb/E2F transcriptional activation pathway.
13
This may
explain the lack of a uniform up-regulation of all APC/C
substrates in some tumor types. Up-regulation of E2F-
mediated transcription due to pRb loss or hyperphospho-
rylation (eg, from cyclin E up-regulation) may dominate
over the effects of altered protein stability due to APC/C
misregulation. Securin and Plk1 are both E2F targets;
therefore, they would tend to cluster together when pRb
transcription repression is misregulated. The effects of
their overexpression on genomic instability would remain
no matter what the mechanism of their misregulation. pRb
is characteristically altered in seminoma, lung cancer,
and transitional cell carcinoma. This would explain in-
creased expression of Emi1 and/or Plk1 and securin in
these tumors without concomitant aurora or Skp2 over-
expression (Figure 4). It is important to note that overex-
pression of a single oncogenic APC/C substrate, such as
aurora A, is sufficient to cause chromosomal instability or
morphological transformation in vitro.
5–9
Another parameter that might explain some of the non-
uniformity of the APC/C cluster data are the inherent
limitations of immunohistochemical analysis. Immunohis-
tochemistry detects various thresholds of protein expres-
sion for different markers due to a variety of factors,
including differences in expression ranges for different
proteins, differences in the efficiencies of primary anti-
bodies to bind target proteins, and differences in efficien-
cies of detecting target proteins in different cellular com-
partments (nucleus versus cytoplasm) in formalin-fixed,
paraffin-embedded tissues.
26
Our data also support the incidence of overexpression
of each APC/C substrate as a strong predictor of malig-
nancy, whereas the absence of overexpression of APC/C
substrates in most cases correlates with benign lesions.
Using nearest shrunken centroids analysis,
25
the predic
-
tive value of having a single APC/C marker up-regulated
is only slightly improved by including additional markers
(Supplemental Figure 4, see http://ajp.amjpathol.org).
This analysis suggests that misregulation of APC/C sub-
strate accumulation is a fairly uniform program down-
stream of APC/C misregulation and that Emi1 overex-
pression is linked to a high percentage of APC/C
substrate-positive tumors.
Within specific tumors, varying percentages of tu-
mors are APC/C substrate positive but Emi1 negative,
including a percentage of neural tumors, gastrointes-
tinal tumors, breast cancers, primitive differentiating
embryonic tumors, sarcomas, and lymphomas (Figures
4 6). In colon tumors, decreased or absent Emi1 may
be explained by high levels of Plk1
30
and/or
-TrCp,
31
the kinase
15
and SCF substrate adapter
14
that trigger
ubiquitin-dependent destruction of Emi1, respectively.
Accumulation of APC/C substrates such as Skp2, Plk1,
and aurora A is common in colon cancer (Figure 4).
Here, we suspect that another form of APC/C misregu-
lation may be occurring, such as APC/C subunit muta-
tion, alterations of other APC/C regulators including
spindle checkpoint proteins such as Mad2, or direct
transcriptional activation or amplification of specific
APC/C substrates (discussed below).
Activation of the G
1
/S Cyclin/pRb/E2F Pathway
Correlates with Emi1 and APC/C Substrate
Protein Levels in Malignant Tumors
To test our hypothesis that activation of the G
1
/S cyclin
program is linked to oncogenic APC/C substrate positiv-
ity, we examined the status of other proliferation path-
ways including proteins critical for G
1
/S control. Accord
-
ingly, we immunostained TMAs for several regulators of
cellular proliferation including cyclins D and E, phosphor-
ylated pRb, E2F3, p27, Bcl2, c-Myc, and
-catenin (Fig-
ures 5 and 6). Here, we looked for linkages between
these G
1
/S programs and misregulation of the APC/C.
We first considered the status of pRb and its regulatory
partners. Tumors with a high incidence of pRb loss in-
cluding lung and hepatic adenocarcinomas showed a
similarly high incidence of Emi1 overexpression and
APC/C substrate positivity, as did cervical adenocarcino-
mas in which pRb is inactivated by human papillomavirus
E7 protein.
In addition to cyclin D/cdk4/6, cyclin E/cdk2 maintains
pRb phosphorylation in S phase through early mitosis,
32
and cyclin E overexpression can induce chromosome in-
stability, similarly to Emi1.
33
We found that cyclin E expres
-
sion clustered with Emi1 in several malignant tumor types
including ovarian, lung, breast, and bladder cancers; oligo-
dendroglial and meningeal neural tumors; leiomyosarcoma,
rhabdomyosarcoma, and malignant fibrous histiocytoma
connective tissue tumors; and rare lymphomas (Figures 5
and 6). Cyclin E immunopositivity statistically correlated with
Emi1 expression in the breast, connective tissue, and can-
cer TMAs (P 0.001) (Supplemental Table 1, see http://
ajp.amjpathol.org). Likewise, cyclin D1 expression corre-
lated with Emi1 in the breast, connective tissue, and
lymphoma TMAs (P 0.002), and phosphorylated-pRb
correlated with Emi1 in all of four TMAs in which it was
examined (breast, connective tissue, cancer, and neural)
(P 0.001). Tumors with a high incidence of loss of the
cyclin D/cdk4 inhibitor p16, an important inhibitor of pRb
phosphorylation, including melanoma, ovarian clear cell
carcinoma, transitional cell cancer, and head and neck
cancer, also showed strong correlations with Emi1 and
APC/C substrate positivity. Thus, Emi1 protein was strongly
expressed in tumors expected to highly express Emi1
mRNA due to biological alterations leading to increased
pRb phosphorylation, namely increased cyclin D or E ex-
pression, or loss of p16.
Emi1 protein expression also clustered with
-catenin
in a large cross section of tumors (Figures 5 and 6). This
correlation was statistically significant across all of the
TMAs (P 0.001) and was strongest for the cancer
APC/C Substrates Are Overexpressed in Tumors 1799
AJP May 2007, Vol. 170, No. 5
Cyclin D2
Cyclin D1
Cyclin E
Emi1
β-catenin
Skp2
Securin
Cdh1
Cyclin A
Ki67
Bcl2
Plk1
Cyclin B
p27
meningioma | angioblastic
medulloblastoma
schwannoma
schwannoma | acoustic
schwannoma
anaplastic astrocytoma
fibrillary astrocytoma
anaplastic astrocytoma
pilocytic astrocytoma
neurofibroma
neurofibroma
neurofibroma
schwannoma
schwannoma
pilocytic astrocytoma
pilocytic astrocytoma
schwannoma | acoustic
astrocytoma | gemistocytic
schwannoma | acoustic
glioblastoma
schwannoma | acoustic
schwannoma | acoustic
oligodendroglioma
meningioma | secretory
oligodendroglioma
anaplastic oligodendroglioma
anaplastic oligodendroglioma
glioblastoma
glioblastoma
medulloblastoma
PNET
anaplastic oligodendroglioma
medulloblastoma
glioblastoma
PNET
PNET
malignant ependymoma
anaplastic astrocytoma
pilocytic astrocytoma
malignant ganglioglioma
anaplastic astrocytoma
astrocytoma | gemistocytic
oligodendroglioma
glioblastoma
glioblastoma
glioblastoma
glioblastoma
glioblastoma
MPNST
oligodendroglioma
glioblastoma
anaplastic oligodendroglioma
anaplastic oligodendroglioma
oligodendroglioma
oligodendroglioma
anaplastic oligodendroglioma
oligodendroglioma
malignant | meningioma
malignant meningioma
malignant papillary meningioma
malignant meningioma
malignant meningioma
gliosarcoma
malignant | meningioma
choroid plexus papilloma
meningioma | secretory
ATRT
hemangioblastoma
malignant | meningioma
hemangioblastoma
ependymoma | myxopapillary
ependymoma
chordoma
fibrillary astrocytoma
anaplastic oligodendroglioma
oligodendroglioma
oligodendroglioma
oligodendroglioma
fibrillary astrocytoma
oligodendroglioma
oligodendroglioma
hemangiopericytoma
ependymoma
ependymoma
oligodendroglioma
oligodendroglioma
oligodendroglioma
oligodendroglioma
oligodendroglioma
gliosarcoma
oligodendroglioma
oligoastrocytoma
oligodendroglioma
oligodendroglioma
anaplastic oligodendroglioma
meningioma | meningothial
leiomyoscarcoma
medulloblastoma
meningioma | meningothial
hemangioblastoma
meningioma | meningothial
anaplastic astrocytoma
ependymoma
malignant | meningioma
meningioma | meningothial
malignant | meningioma
hemangioblastoma
hemangioblastoma
ependymoma
malignant meningioma
glioblastoma
E2F3
c-Myc
Bcl2
p27
Cyclin A
Ki67
phos-pRb
Skp2
Cyclin E
β-catenin
Aurora A
Emi1
Cyclin D1
Securin
Plk1
Cdh1
Neural TMA
Lymphoma TMA
A
B
Emi1 - / APC Substrate -
Low Grade Neural Tumors:
Neurofibromas, Schwanomas,
Pilocytic Astrocytomas
Few Intermediate Grade &
High GradeTumors
Emi1 - / APC Substrate +
High Grade Malignant Tumors:
Anaplastic Astrocytomas,
Anaplastic Oligodendrogliomas,
Glioblastomas, PNETs,
Meduloblastomas
One Low Grade &
Few Intermediate Grade Tumors
Emi1 + /APC Substrate +
Intermediate Grade Tumors:
Oligodendroglioma,
Ependymoma
High Grade Tumors:
Gliosarcoma, Medulloblastoma,
ATRT, Leiomyosarcoma
Few Low Grade Tumors:
Hemangioblastoma, Meningioma
FL1
FL3
FL1
FL3
FL1
DLBCL
DLBCL
FL3
Burkitt
CLL/SLL
FL1
FL1
FL3
FL2
FL1
FL2
Marginal Zone | splenic
Marginal Zone | splenic
Marginal Zone | splenic
Maginal Zone
Marginal Zone | splenic
CLL/SLL
FL1
CLL/SLL
FL3
FL1
FL1
FL1
FL1
FL1
FL2
FL2
FL1
FL2
FL2
FL1
FL1
FL1
FL2
malignant mast cell
malignant mast cell
DLBCL
FL3
FL3
Mantle Cell
Mantle Cell
Mantle Cell
Mantle Cell
Mantle Cell
Hodgkin | mixed cell
lymphoblastic T-cell
lymphoblastic T-cell
FL3
FL3
DLBCL
FL3
DLCL
DLBCL
FL3
FL3
DLBCL
DLBCL
DLBCL
Mantle Cell | colon
FL3
DLBCL
FL2
DLBCL
DLBCL
DLBCL | small bowel
Marginal Zone | extranodal
FL3
FL1 | soft tissue
DLBCL | tonsil
lymphoblastic T-cell | skin
Marginal Zone | extranodal
FL3
FL2
FL3
FL2
FL2
FL3
peripheral T-cell
FL3
lymphoblastic T-cell
lymphoblastic T-cell
DLBCL | parotid
DLBCL
Maliginant Histiocytosis
DLBCL | lacrimal sac
lymphoblastic T-cell
DLBCL
PTLD | NKC | small bowel
DLBCL
malignant NKC | stomach
Hodgkin | NLP
Hodgkin | NLP
FL3
DLCL
FL3
multiple myeloma
FL3
FL3
CLLSLL
CLLSLL
FL1
FL1
FL1
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
Marginal Zone | extranodal
FL1
peripheral T-cell
DLBCL
PTLD | malignant B cell
Marginal Zone | extranodal
FL3
LPL
peripheral T-cell
DLBCL T-cell rich
FL3
DLBCL T-cell rich
FL2
DLBCL
FL3
DLBCL
DLBCL
DLBCL
DLBCL
DLBCL
anaplastic large cell
NKC | skin
peripheral T-cell
anaplastic large cell
peripheral T-cell
Marginal Zone | extranodal
peripheral T-cell
FL3
peripheral T-cell | Lennert’s
anaplastic large cell
Emi1- /APC Substrate -
Low Grade Lymphomas:
Folicular Lymphoma Grade I
Splenic Marginal Zone
CLL/SLL
Few High Grade Lymphomas
Emi1- /APC Substrate +
Intermediate Grade Lymphomas:
Folicular Grade II, Mantle Cell
High Grade Lymphomas:
Folicular Grade III, DLBCL
Few Low Grade Lymphomas
Emi1 + /APC Substrate +
Mostly High Grade Lymphomas:
Folicular Grade III, DLBCL,
Peripheral T Cell,
Lymphoblastic T Cell
Some Intermediate Grade:
Extranodal Marginal Zone,
Folicular Grade II
Few Low Grade Lymphomas
1800 Lehman et al
AJP May 2007, Vol. 170, No. 5
and connective tissue tumor arrays (P 0.05 and
0.001, respectively; Supplemental Table 1, see http://ajp.
amjpathol.org). Wnt signals through
-catenin to induce S
phase.
34
Interestingly, both Emi1 and
-catenin are reg
-
ulated by
-TrCP, which can restrain cell-nonautonomous
signaling through the Wnt and Hedgehog pathways
35
as
well as the cell autonomous overduplication of centro-
somes with overexpression of Emi1.
14
APC/C Mitotic Substrate Misregulation Is
Distinct from Up-Regulation of Proliferation
Proteins and Is a Marker of Aggressive Tumors
In many tissue types, including neural and connective
tissue tumors, lymphomas, and a subset of carcinomas,
general S phase markers including Ki67, cyclin A, Bcl2,
phosphorylated-pRb, and E2F3 form a “proliferation”
cluster distinct from that containing the mitotic regulatory
and oncogenic APC/C substrates securin, aurora A, Plk1,
Skp2, and the APC/C-activating subunit Cdh1, “mitotic
APC/C cluster.” With few exceptions, proliferative but
benign World Health Organization grade I neural tumors,
such as neurofibromas, schwannomas, and pilocytic as-
trocytomas, expressed the proliferation cluster but were
generally negative for the APC/C cluster, whereas malig-
nant tumors, such as grade II–IV gliomas, were nearly
uniformly immunopositive for both clusters (Figure 5A).
This trend also occurred for lymphoid and connective
tissue tumors. World Health Organization grade I to II
follicular lymphomas are generally proliferation cluster
positive and APC/C cluster negative, whereas grade III
follicular and diffuse large B-cell lymphomas are immu-
nopositive for both clusters (Figure 5B). In addition, all of
the diffuse large B-cell lymphomas expressing cyclin D2,
a marker of poor prognosis,
36
were in the Emi1-positive
subset. Benign connective tissue tumors are generally
Emi1, securin, and Skp2 immunonegative. Malignant
connective tissue tumors (sarcomas) are only occasion-
ally Emi1 immunopositive but frequently highly express
securin and Skp2 (Figure 6).
In contrast to most carcinomas, prostate and thyroid
tumors, which are usually less aggressive cancers, were
often mitotic APC/C mitotic cluster negative (Figure 4).
Thus in some cases, overexpression of Emi1 and in many
cases, overexpression of the oncogenic mitotic control
APC/C substrates seem to be markers of tumor
aggressiveness.
A high percentage of malignant tumors appeared to
markedly accumulate the APC/C activator subunit Cdh1,
whereas most benign and some low-grade tumors were
Cdh1 immunonegative (Figure 4). This suggests that Cdh1
overexpression may be a response to APC/C inhibition.
Cdh1 overexpression in tumors with reduced APC/C activity
could represent a compensatory feedback loop. It may
seem counterintuitive that both the APC/C activator Cdh1
and APC/C inhibitor Emi1 are concomitantly overexpressed
in some tumor types; however, this is not surprising when
one considers that Cdh1 is itself an APC/C substrate
17
and
that Emi1 knockdown by siRNA decreases Cdh1 levels
(N.L.L., unpublished data). Furthermore, Emi1 overexpres-
sion can override the effects of Cdh1 overexpression in
vitro,
13
possibly because of stabilization of cyclin A, be
-
cause Cdh1 is inactivated by phosphorylation via cyclin
A/cdk.
37
Specifically, Emi1 overexpression has been shown
to relieve the transient cell cycle block caused by Cdh1
overexpression.
13
Again, this is not unexpected because
Emi1 can bind either free Cdh1 or the APC/C core subunit
complex and block APC/C activity.
12
Cdh1 overexpression may thus reflect an imbalance of
positive and negative APC/C regulation in tumors. Nota-
bly, the APC/C activator Cdc20 has been reported to be
overexpressed at the mRNA level in gastric and lung
cancers.
38,39
Although Cdh1 normally acts to induce cell
cycle exit and thus limit cell cycle progression, Cdh1
overexpression can result in massive over-replication of
the genome
40
; therefore Cdh1 overexpression could lead
to genomic instability in some circumstances.
In some tumor types, particularly in mesoderm-derived
tissue tumors, Plk1 seems to be broadly expressed in
both malignant and benign neoplasms (Figure 4). Be-
cause Plk1 is an E2F target gene
41
that is involved in
numerous phosphorylation reactions during G2 and mi-
tosis,
42
it may thus be more of a general marker of pro
-
liferation, perhaps specifically in hematolymphoid and
connective tissue (mesoderm-derived) tumors. In sup-
port of this, we found that Plk1 protein expression
strongly correlated with phosphorylated pRb in the con-
nective tissue and cancer TMAs with correlation coeffi-
cients of 0.267 (P 0.000) and 0.440 (P 0.000),
respectively (Supplemental Table 1, see http://ajp.
amjpathol.org). In contrast to mesoderm-derived tumors,
Plk1 expression correlated with increasing histopatholog-
ical grade in colon adenocarcinomas and neuroecto-
derm-derived tumors (Figure 4).
The timely destruction of cyclins and other central
mitotic regulators by the APC/C is essential to the accu-
rate segregation of chromosomes and the maintenance
of genomic stability. Several mechanisms may lead to
Figure 5. Hierarchical clustering analysis of Emi1, proliferation control, and mitotic control proteins in human tumors. For each tumor type, immunopositivity for
Emi1, proliferation control (cyclins D1, D2, E, and A;
-catenin; p27
Kip1
; Bcl2; and Ki67), and mitotic control/APC/C (aurora A, Plk1, securin, Cdh1, and Skp2)
substrates were identified. Dendrograms for neural tumor (A) and lymphoma (B) TMAs are shown. Green rectangles indicate a tumor showing no immuno-
positivity, dark red indicates moderate positivity (3 to 29% of tumor cells positive), and bright red indicates high positivity (30% tumor cells positive). Gray
rectangles indicate unscorable TMA cores. The dendograms on the top horizontal axes show clusters of proliferation control proteins and APC/C substrate
proteins. Oncogenic APC/C substrates securin, aurora A, Plk1, and Skp2 (red) form a cluster with Emi1 distinct from broader proliferation markers such as Ki67,
cyclin A, and Bcl-2 in malignant neural tumors and lymphomas. The pink rectangles on the right vertical axes represent Emi1-positive tumors, which are largely
malignant and positive for both proliferation and APC/C clusters. The yellow rectangles indicate mostly malignant tumors that are largely prolifer ation and APC/C
cluster positive but Emi1 negative. The blue rectangles represent mostly benign or low-grade tumors that are predominantly proliferation cluster positive and
APC/C cluster negative. ALCL, anaplastic large cell lymphoma; DLBCL, diffuse large B-cell lymphoma; FL1–3, follicular lymphoma grades 1 through 3; NKC, natural
killer cell (lymphoma); NLP, nodular lymphocyte predominant Hodgkin’s lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma;
PTLD, posttransplant lymphoproliferative disorder.
APC/C Substrates Are Overexpressed in Tumors 1801
AJP May 2007, Vol. 170, No. 5
benign | atypical lipoma
benign | hemangioendothelioma | epithelioid
benign | DFSP
benign | leiomyoma
benign | leiomyoma
malignant | fibromyosarcoma | metastatic
benign | schwannoma
malignant | MFH
malignant | leiomyosarcoma
benign | fasciitis | nodular
malignant | MPNST
benign | atypical lipoma
benign | fibromatosis
benign | fibromatosis
malignant | DSRCT
malignant | MPNST
malignant | MPNST
benign | atypical lipoma
malignant | liposarcoma | pleomorphic
malignant | myosarcoma
malignant | synovial sarcoma
malignant | angiosarcoma
benign | DFSP
malignant | angiosarcoma
malignant | giant cell tumor | tenosynovial
benign | fibroanthoma | atypical
malignant | synovial sarcoma
malignant | GIST
malignant | carcinosarcoma
malignant | MFH
malignant | leiomyosarcoma
malignant | GIST
malignant | GIST
malignant | GIST
malignant | leiomyosarcoma
malignant | carcinosarcoma
malignant | carcinosarcoma | metastatic
malignant | MFH
malignant | osteosarcoma
malignant | MFH
malignant | clear cell sarcoma
malignant | carcinosarcoma | recurrent
malignant | liposarcoma | dedifferentiated
malignant | sarcoma | GIST
malignant | GIST
malignant | PNET
malignant | MFH
malignant | leiomyosarcoma
malignant | liposarcoma | dedifferentiated
malignant | SFT
malignant | rhabdomyosarcoma
malignant | osteosarcoma
malignant | osteosarcoma
malignant | leiomyosarcoma
malignant | rhabdomyosarcoma
malignant | osteosarcoma
malignant | SFT
malignant | sarcoma | NOS
malignant | liposarcoma | dedifferentiated
malignant | MPNST
malignant | MFH
malignant | MFH
malignant | osteosarcoma
malignant | SFT
malignant | SFT
malignant | SFT
malignant | osteosarcoma
malignant | SFT
malignant | synovial sarcoma
malignant | liposarcoma | dedifferentiated
malignant | MFH
malignant | clear cell sarcoma
malignant | angiosarcoma
malignant | synovial sarcoma
malignant | leiomyosarcoma
malignant | giant cell tumor | tenosynovial
malignant | GIST
malignant | giant cell tumor | tenosynovial
malignant | synovial sarcoma
malignant | MFH
malignant | endometrial sarcoma
malignant | MFH
malignant | adenosarcoma
malignant | MFH
malignant | MFH
malignant | rhabdomyosarcoma | metastatic
malignant | MFH
malignant | MFH
malignant | MFH
malignant | MFH
malignant | MPNST
malignant | MPNST
malignant | sarcoma | angiosarcoma
malignant | giant cell tumor | tenosynovial
benign | fibroanthoma
benign | fibroanthoma
malignant | lipomatous tumor | liposarcoma
malignant | epithelioid sarcoma
malignant | fibrosarcoma
benign | lipoblastomatosis
malignant | liposarcoma
benign | lipoblastomatosis
malignant | MPNST
malignant | Ewing’s sarcoma
malignant | rhabdomyosarcoma
malignant | rhabdomyosarcoma
malignant | carcinosarcoma
malignant | Ewing’s sarcoma
malignant | Ewing’s sarcoma
malignant | rhabdomyosarcoma
malignant | liposarcoma
malignant | leiomyosarcoma
malignant | rhabdomyosarcoma | metastatic
malignant | endometrial stromal sarcoma
malignant | rhabdomyosarcoma
malignant | MFH
malignant | leiomyosarcoma
malignant | MFH
malignant | GIST
malignant | carcinosarcoma
malignant | leiomyosarcoma | recurrent
malignant | GIST
benign | glomus tumor
malignant | sarcoma | leiomyosarcoma
malignant | GIST
benign | glomus tumor
benign | hemangioendothelioma
benign | hemangioendothelioma | infantile
benign | hemangioendothelioma
benign | glomus tumor
benign | atypical lipoma
malignant | DRSCT
malignant | rhabdomyoma
malignant | MFH
benign | fibromatosis
benign | fibroanthoma
malignant | fibrosarcoma
malignant | leiomyosarcoma
malignant | alveolar soft part sarcoma
malignant | Ewing’s sarcoma
malignant | Ewing’s sarcoma
malignant | chondrosarcoma | myoid
malignant | fibrosarcoma
benign | leiomyoma
malignant | endometrial stromal sarcoma
benign | glomus tumor
malignant | MPNST
malignant | Ewing’s sarcoma
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | MFH
malignant | leiomyosarcoma | epitheliod
malignant | leiomyosarcoma
malignant | leiomyosarcoma
malignant | clear cell sarcoma
benign | DFSP
malignant | osteosarcoma
malignant | chondrosarcoma
malignant | MFH
malignant | rhabdomyosarcoma
malignant | rhabdomyosarcoma
malignant | carcinosarcoma
malignant | MPNST
malignant | rhabdomyosarcoma
malignant | Ewing’s sarcoma
malignant | MFH
malignant | osteosarcoma
malignant | leiomyosarcoma
malignant | MFH
malignant | MFH
malignant | MFH
malignant | MFH
Skp2
Securin
pho
s
-pRb
Cyclin
E
cycl
in D1
Plk1
p27
Cyclin A
Cdh1
Skp2
Securin
phos-pRb
Em
i
1
Cyclin E
cyclin D1
Plk1
p27
Cyclin A
Cdh1
Connective Tissue
Tumor TMA
malignant | MFH
Emi1 - / APC -
Mostly Benign
Emi1 + / APC+
Mostly Malignant
Emi1 - / APC +
Mostly Malignant
Emi1
Figure 6. Hierarchical clustering analysis of Emi1, proliferation control, and mitotic control proteins in human connective tissue tumors. Data are represented as
in Figure 4. DFSP, dermatofibrosarcoma protuberans; DSRCT, desmoplastic small round cell tumor; GIST, gastrointestinal stromal cell tumor; MFH, malignant
fibrous histiocytoma; MPNST, malignant peripheral nerve sheath tumor; NOS, type not otherwise specified; PNET, primitive neuroectodermal tumor; SFT, solitary
fibrous tumor.
1802 Lehman et al
AJP May 2007, Vol. 170, No. 5
APC/C misregulation in tumors including inactivating mu-
tations or down-regulation of APC/C subunits
43,44
; muta
-
tion, loss, or overexpression of spindle checkpoint pro-
teins such as Mad2,
18,20
Bub1,
45
or BubR1
46,47
;
overexpression of Emi1; or inactivation of the potential
APC/C inhibitor RASSF1A.
48
In addition, some APC/C
substrates may themselves be misexpressed through
genomic alterations, eg, aurora A gene amplification in
breast, gastric and colon cancers.
49
Because Plk1 and
securin are E2F transcriptional targets,
41,50
they can also
be overexpressed at the mRNA level by pRb misregula-
tion or loss.
A small number of studies to date have suggested that
specific substrates of the APC/C are misregulated in certain
tumors.
5–7,50 –56
However, no general survey of the extent or
uniformity of APC/C substrate misregulation in all classes of
human tumors has been published. Our analysis of more
than 1600 tumor samples, representing more than 80 tumor
classes, provides the first systematic analysis of APC/C
substrate misregulation. We found that protein levels of
mitotic APC/C substrates are frequently and coordinately
elevated in malignant human tumors, in many cases with
concomitant overexpression of the APC/C inhibitor Emi1.
Overaccumulation of APC/C substrates could be, in some
cases, directly because of their increased transcription. In
other cases, inappropriate protein stability, secondary to
APC/C misregulation by APC/C mutations, Emi1, or Mad2
misexpression may be an important primary or contributing
factor. The latter is supported by the ability of overex-
pressed Emi1 and Mad2 to stabilize APC/C substrates and
cause genomic instability in vitro.
20 –21
In addition, because
APC/C substrates are substantially regulated by ubiquitin-
dependent proteolysis, it would not be unexpected that
altered degradation plays a role in their overexpression in
neoplasia.
Our analysis demonstrates a strong correlation between
APC/C misregulation and malignancy and an anti-correla-
tion with nonmalignancy. This profile is independent of mis-
regulation of G
1
/S phase cell cycle markers (cyclin A, Ki67,
and E2F), which are also strongly accumulated in benign
tumors. Importantly, in malignant tumors, both the G
1
/S
phase cell cycle markers and the APC/C markers are cor-
related. This distinction between the prevalence of G
1
/S
phase cell cycle misregulation and mitotic APC/C substrate
misregulation suggests several conclusions. First, it sug-
gests that G
1
/S phase cell cycle regulation, presumably
linked to increased proliferation, is representative of a
broader class of hyperproliferative processes and may be
linked to the activation of growth signaling pathways. Sec-
ond, it suggests that the misregulation of mitotic APC/C
substrates occurs through an independent mechanism, not
strictly linked to G
1
/S phase control. A strong candidate for
this independent mechanism is the stabilization control
pathway regulated by APC/C inhibitors including Emi1 and
Mad2. Our present data support that Emi1 overexpression
is linked to a sizable fraction of cases where mitotic APC/C
substrates are misregulated in tumors but not to all cases.
Functional tests from our laboratory show that overexpres-
sion of Emi1 is sufficient to stabilize APC/C substrates,
creating a state much like that seen in tumors.
14,21
For those
tumors lacking Emi1 overexpression, it may be that 1) Emi1
is reduced by loss of heterozygosity following an event of
genomic instability, 2) other APC/C regulators such as
Mad2 can direct APC/C misregulation, or 3) independent
control mechanisms determine the increased accumulation
of these mitotic regulators. Additional studies to look at
Mad2 in tumors are ongoing.
Besides Mad2, misexpression or mutation of other
spindle checkpoint proteins, such as BubR1, may be
linked to genomic instability. BubR1 is necessary for
apoptosis after prolonged spindle damage and is signif-
icantly decreased in approximately 30% of colon adeno-
carcinomas,
46
and biallelic mutations in BUB1B, which
encodes BubR1, have recently been reported in families
affected by Mosaic Variegated Aneuploidy syndrome,
which manifests in mosaic aneuploidy and predisposition
to childhood malignancies.
47
In addition to Emi1 misregulation, misregulation of
other APC/C regulators that are E2F targets, such as
Mad2 and BubR1, and/or E2F-regulated APC/C sub-
strates, such as securin or Plk1, could be contributing or
alternate mechanisms of mitotic APC/C substrate mis-
regulation in cancers, independent of Emi1. Thus, both
increased E2F-mediated transcription and inappropriate
protein stability probably work in concert in the path-
way to genomic instability downstream of pRb/E2F
misregulation.
We observed that an increase in the protein expression
levels of the mitotic control APC/C substrates Skp2, securin,
aurora A, and Plk1; the APC/C regulator Cdh1; and in many
cases the APC/C inhibitor Emi1, strongly correlates with
malignancy. Furthermore, in some tumors, such as lympho-
mas, increased accumulation of these proteins may corre-
late with tumor aggressiveness. We suggest that this is due
to an increased propensity for mitotic catastrophe and
genomic instability. However, despite the in vitro data show-
ing that overexpression of Emi1 and APC/C substrates re-
sults in chromosomal instability, the ultimate biological con-
sequences of APC/C substrate overexpression in tumors
are unclear. The question of whether APC/C substrate over-
expression is causal or only a downstream consequence of
tumorigenesis and genomic instability cannot be answered
by the present study.
Nevertheless, the observation of up-regulation of the
APC/C pathway in tumors could prove to be of clinical
importance. The pathway may be therapeutically exploit-
able through pharmacological targeting of Emi1 or other
APC/C regulators or substrates. The propensity for
genomic instability in tumors may also be related to sen-
sitivity to antimitotics; APC/C substrate profiles might
serve as a predictive marker for cancers responsive or
resistant to agents that target the spindle checkpoint, eg,
taxanes,
52
or other regulators of mitosis. Last, because
the mitotic APC/C expression profile not only distin-
guishes nonmalignant from malignant proliferations in
certain tumor classes, such as connective tissue neo-
plasms and a large subset of neural tumors, but also is
nearly uniform in certain specific tumor types, such as
seminomas and clear cell carcinomas of the ovary, indi-
vidual APC/C substrates and/or Emi1 may have broader
utility in diagnostic pathology. The near uniformity of
Cdh1 overexpression in malignant versus benign tumors
APC/C Substrates Are Overexpressed in Tumors 1803
AJP May 2007, Vol. 170, No. 5
may prove to be a valuable diagnostic tool. Further stud-
ies will clearly be necessary to explore these important
clinical possibilities.
Acknowledgments
We thank Dr. Kenneth Ban for assistance with siRNA, Dr.
Julie D. Reimann for assistance early in the study, and
Elizabeth Domanay and Shuchun Zhao for technical help.
We thank Dr. Peter R. Egbert for providing retinoblastoma
cases, Dr. Teri A. Longacre for providing ovarian tumor
samples, Dr. Claude Prigent for anti-aurora A antibody,
and Caroline Tudor and Anet James for assistance with
digital photo editing. We also thank Dr. Michael L. Cleary
and Dr. Andrew J. Connolly for invaluable comments.
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APC/C Substrates Are Overexpressed in Tumors 1805
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    • "For example, EMI1 is significantly overexpressed in ovarian tumors and its overexpression correlates positively with high histological grade and poor patient survival [88,89]. Similarly, EMI1 overexpression was noted in a number of malignant tumors when compared to benign tumors [90] . Experimental evidence supporting an oncogenic role for EMI1 comes from the observations that its overexpression enhances proliferation and genomic instability in p53-deficient cells [128], and enhances the proliferation of chronic myeloid leukemia cells expressing the BRC-ABL fusion oncopro- tein [129]. "
    [Show abstract] [Hide abstract] ABSTRACT: F-box proteins are substrate receptors of the SCF (SKP1-Cullin 1-F-box protein) E3 ubiquitin ligase that play important roles in a number of physiological processes and activities. Through their ability to assemble distinct E3 ubiquitin ligases and target key regulators of cellular activities for ubiquitylation and degradation, this versatile group of proteins is able to regulate the abundance of cellular proteins whose deregulated expression or activity contributes to disease. In this review, we describe the important roles of select F-box proteins in regulating cellular activities, the perturbation of which contributes to the initiation and progression of a number of human malignancies.
    Full-text · Article · Oct 2015
    • "These findings indicate a broader CARP-1/CCAR1 association with APC/C proteome [61]. In light of the fact that APC/C plays a distinct role in various cell cycle checkpoints [62, 63] and deregulation of APC/C and its regulators and substrates has been implicated in tumor progression [64], the components of the APC/C proteome including its co-activator CARP-1/ CCAR1 therefore represent attractive targets for design of cell cycle inhibitory strategies with potential for therapeutic use656667. "
    [Show abstract] [Hide abstract] ABSTRACT: Targeted cancer therapy using small molecule inhibitors (SMIs) has been useful in targeting the tumor cells while sparing the normal cells. Despite clinical success of many targeted therapies, their off-target effects and development of resistance are emerging as significant and challenging problems. Thus, there is an urgent need to identify targets to devise new means to treat cancers and their drug-resistant phenotypes. CARP-1/CCAR1 (Cell division cycle and apoptosis regulator 1), a peri-nuclear phospho-protein, plays a dynamic role in regulating cell growth and apoptosis by serving as a co-activator of steroid/thyroid nuclear receptors, β-catenin, Anaphase Promoting Complex/Cyclosome (APC/C) E3 ligase, and tumor suppressor p53. CARP-1/CCAR1 also regulates chemotherapy-dependent apoptosis. CARP-1/CCAR1 functional mimetics (CFMs) are a novel SMIs of CARP-1/CCAR1 interaction with APC/C. CFMs promote apoptosis in a manner independent of p53. CFMs are potent inhibitors of a variety of cancer cells including the drug (Adriamycin or Tamoxifen)-resistant breast cancer cells but not the immortalized breast epithelial cells, while a nano-lipid formulation of the lead compound CFM-4 improves its bioavailability and efficacy in vivo when administered orally. This review focuses on the background and pleiotropic roles of CARP-1/CCAR1 as well as its apoptosis signaling mechanisms in response to chemotherapy in cancer cells.
    Full-text · Article · Mar 2015
    • "APC/C is a multi-subunit ubiquitin E3 ligase protein that plays a distinct role in cell cycle transitions [11], [12]. Previous studies showed that misregulation of APC/C and its substrates correlates with tumor progression [13]. We identified a novel class of small molecule inhibitors (SMIs) of CARP-1 binding with APC/C subunit APC2. "
    [Show abstract] [Hide abstract] ABSTRACT: Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and β proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents.
    Full-text · Article · Jul 2014
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