Aurora-A Expression Is
with Chromosomal Instability
in Colorectal Cancer1
Yoshifumi Baba*,2, Katsuhiko Nosho*,2,
Kaori Shima*,2, Natsumi Irahara*, Shoko Kure*,
Saori Toyoda*, Gregory J. Kirkner†, Ajay Goel‡,
Charles S. Fuchs*,†and Shuji Ogino*,§,¶
*Department of Medical Oncology, Dana-Farber Cancer
Institute and Harvard Medical School, Boston, MA, USA;
†Channing Laboratory, Department of Medicine, Brigham and
Women’s Hospital and Harvard Medical School, Boston, MA,
USA;‡Department of Internal Medicine, Baylor University
Medical Center, Dallas, TX, USA;§Department of Pathology,
Brigham and Women’s Hospital and Harvard Medical School,
Boston, MA, USA;¶Department of Epidemiology, Harvard
School of Public Health, Boston, MA, USA
AURKA(theofficialsymbol for Aurora-A,STK15,orBTAK)regulates the functionofcentrosomes,spindles,and kinetochores
for proper mitotic progression. AURKA overexpression is observed in various cancers including colon cancer, and a link
between AURKA and chromosomal instability (CIN) has been proposed. However, no study has comprehensively exam-
ined AURKA expression in relation to CIN or prognosis using a large number of tumors. Using 517 colorectal cancers in
two prospective cohort studies, we detected AURKA overexpression (by immunohistochemistry) in 98 tumors (19%). We
assessed other molecular events including loss of heterozygosity (LOH) in 2p, 5q, 17q, and 18q, the CpG island
methylation phenotype (CIMP), and microsatellite instability (MSI). Prognostic significance of AURKA was evaluated by
Cox regression and Kaplan-Meier method. In both univariate and multivariate logistic regressions, AURKA overexpression
was significantly associated with CIN (defined as the presence of LOH in any of the chromosomal segments; multivariate
odds ratio, 2.97; 95% confidence interval, 1.40-6.29; P = .0045). In multivariate analysis, AURKA was associated with cyclin
history of colorectal cancer (P = .050), but not with sex, age, body mass index, tumor location, stage, CIMP, MSI, KRAS,
BRAF,BMI,LINE-1hypomethylation,p53,p21,β-catenin,orcyclooxygenase 2.AURKA was notsignificantlyassociatedwith
clinical outcome or survival. In conclusion, AURKA overexpression is independently associated with CIN in colorectal can-
cer, supporting a potential role of Aurora kinase-A in colorectal carcinogenesis through genomic instability (rather than epi-
Neoplasia (2009) 11, 418–425
Abbreviations: AURKA, Aurora-A; BMI, body mass index; CI, confidence interval; CIN, chromosomal instability; CIMP, CpG island methylator phenotype; FASN, fatty acid
synthase; HPFS, Health Professionals Follow-up Study; HR, hazard ratio; LOH, loss of heterozygosity; MSI, microsatellite instability; MSS, microsatellite stable; NHS, Nurses’
Health Study; OR, odds ratio
Address all correspondence to: Shuji Ogino, MD, PhD, Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical
School, 44 Binney St, Room JF-215C, Boston, MA 02115. E-mail: firstname.lastname@example.org
1This work was supported by US National Institutes of Health (NIH) grants P01 CA87969, P01 CA55075, P50 CA127003 (to C.S.F.), and K07 CA122826 (to S.O.) and in part by
grants from the Bennett Family Fund and from the Entertainment Industry Foundation through the National Colorectal Cancer Research Alliance (NCCRA). K.N. was supported by a
fellowshipgrantfromthe JapanSociety for PromotionofScience.Thecontent issolely theresponsibilityofthe authorsanddoesnotnecessarilyrepresentthe officialviews of theNational
Cancer Institute or NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No conflicts of interest exist.
2YB, KN, and KS contributed equally.
Received 13 January 2009; Revised 16 February 2009; Accepted 16 February 2009
Copyright © 2009 Neoplasia Press, Inc. All rights reserved 1522-8002/09/$25.00
Volume 11 Number 5May 2009pp. 418–425
Chromosomal instability (CIN) in cancer is characterized by frequent
chromosomal abnormalities including translocations, gains, and losses
of chromosomes or their segments. Chromosomal instability promotes
carcinogenesis through loss of tumor suppressors and copy number
gains of oncogenes . Causes of CIN are still poorly understood
but possibly include mitotic spindle checkpoint gene (e.g., BUB1
and BUB1B) deregulation , DNA checkpoint gene (e.g., TP53)
mutation , cell cycle regulator (e.g., FBXW7) inactivation ,
telomere dysfunction , and abnormal centrosome number and
function [6–8]. Centrosome dysfunction causes abnormal centrosome
segregation during mitosis, which may lead to CIN [6–8].
AURKA (the official symbol for Aurora-A, also known as STK15/
BTAK) is a member of the Aurora family of cell cycle–regulating
serine/threonine kinases and functions in centrosome regulation
and mitotic spindle formation [9,10]. Activation of AURKA in ex-
perimental systems confers malignant phenotype by inducing centro-
some amplification and genomic instability, indicating AURKA as an
oncogene [11–13]. AURKA expression has been reported in various
human cancers [14–20], including colorectal cancers . AURKA
amplification correlates with CIN in colorectal cancer . However,
to our knowledge, no study has comprehensively examined the rela-
tion between AURKA protein overexpression and CIN in colorectal
cancer or whether the relation is independent of other related molecu-
lar features including microsatellite instability (MSI) and the CpG
island methylator phenotype (CIMP).
In this study using a large number (N = 517) of colorectal cancers,
we examined AURKA overexpression in relation to CIN and patient
survival. Because our tumor database included other related molecular
events, we were able to assess whether there was an independent rela-
tion between AURKA and CIN. Our current data support AURKA as
one of potential contributing factors for CIN in colorectal cancer.
Materials and Methods
We used the databases of two large prospective cohort studies; the
Nurses’ Health Study (NHS; N = 121,700 women followed since
1976) [22,23], and the Health Professionals Follow-up Study
(HPFS; N = 51,500 men followed since 1986) . Data on height
and weight were obtained by biennial questionnaire. A subset of the
cohort participants developed colorectal cancers during prospective
follow-up. Previous studies on the NHS and HPFS have described
baseline characteristics of cohort participants and incident colorectal
cancer cases and confirmed that our colorectal cancers were good
representatives of a population-based sample [22,23]. Data on tumor
location and stage were obtained through medical record review.
We collected paraffin-embedded tissue blocks from hospitals where
patients had undergone resections of colorectal cancers. On the basis
of availability of adequate tissue specimens and results, a total of
517 colorectal cancers were included. Written informed consent
was obtained from all study subjects. Among our cohort studies,
there was no significant difference in demographic features between
cases with tissue available and those without available tissue .
This current analysis represents a new analysis of AURKA on the
existing colorectal cancer database that have been previously charac-
terized for CIMP, MSI, p53, KRAS, BRAF, PIK3CA, long interspersed
nucleotide element 1 (LINE-1), cyclooxygenase 2 (COX-2), and clini-
cal outcome [23–26], which is analogous to novel studies using the
well-described cell lines or animal models. In any of our previous stud-
ies, we have not examined AURKA expression or the relations of
AURKA with clinical outcome and other molecular events. This study
represents a unique novel study in term of 1) a large sample size
analyzed for AURKA; 2) the comprehensive clinical and tissue molec-
ular database used, including the long-term follow-up outcome data,
CIMP, MSI, KRAS, BRAF, PIK3CA, p53, β-catenin, LINE-1 methyla-
tion, and COX-2; and 3) a number of molecular correlates that have
been analyzed. Tissue collection and analyses were approved by the
Harvard School of Public Health and Brigham and Women’s Hospital
Institutional Review Boards.
Hematoxylin and eosin–stained tissue sections were examined by a
pathologist (S.O.) unaware of other data. The tumor grade was cate-
gorized as low (≥50% gland formation) versus high (<50% gland
formation). The presence and extent of extracellular mucin were cate-
gorized as 0% (no mucin), 1% to 49%, or ≥50% of the tumor volume
. The presence and extent of signet ring cells were categorized as
absent (0%) or present (≥1%) .
Sequencing of KRAS, BRAF, and PIK3CA
Genomic DNA was extracted from tumor and polymerase chain
reaction (PCR) and Pyrosequencing targeted for KRAS (codons 12 and
13) , BRAF (codon 600) , and PIK3CA (exons 9 and 20) 
were performed as previously described.
Microsatellite Instability Analysis
Microsatellite instability analysis was performed, using 10 micro-
satellite markers (D2S123, D5S346, D17S250, BAT25, BAT26,
BAT40, D18S55, D18S56, D18S67, and D18S487) . MSI-high
was defined as the presence of instability in ≥30% of the markers.
MSI-low was defined as instability in <30% of the markers, and micro-
satellite stable (MSS) tumors were defined as tumors without an un-
stable marker .
Loss of Heterozygosity Analysis
For loss of heterozygosity (LOH) analysis using microsatellite markers
(D2S123, D5S346, D17S250, D18S55, D18S56, D18S67, and
D18S487), we duplicated PCR in each sample to exclude allele drop-
outs of one of two alleles [27,31]. Loss of heterozygosity at each locus
was defined as ≥40% reduction of one of two allele peaks in tumor
DNA relative to normal DNA. Chromosomal instability (CIN) pos-
itivity was defined as the presence of LOH in any of the chromosomal
segments among 2p, 5q, 17q, and 18q. CIN negativity was defined as
the absence of LOH in any of the chromosomal segments with the
presence of at least two informative segments.
Real-time PCR for Quantitative DNA Methylation Analysis
Sodium bisulfite treatment on genomic DNA and subsequent real-
time PCR (MethyLight) were validated and performed as previously
promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1,
NEUROG1, RUNX3, and SOCS1] [24,33,34]. CIMP-high was de-
fined as the presence of six or more of eight methylated promoters,
CIMP-low as the presence of one to five of eight methylated promoters,
Neoplasia Vol. 11, No. 5, 2009AURKA and CIN in Colorectal Cancer Baba et al.
and CIMP-0 as the absence of methylated promoters, according to the
previously established criteria [24,35].
Pyrosequencing to Measure LINE-1 Methylation
To accurately quantify relatively high methylation levels in
LINE-1 repetitive elements, we used Pyrosequencing as previously
Immunohistochemistry for AURKA, p53, p21, Cyclin D1,
β-Catenin, COX-2, and Fatty Acid Synthase
Tissue microarrays were constructed as previously described [23,37].
Methods of immunohistochemical procedures and examples of staining
patterns can be found in the previous reports as follows: p53 , p21
(CDKN1A) [39,40], cyclin D1 , β-catenin , COX-2 [27,43],
and fatty acid synthase (FASN) [27,44]. For AURKA, antigen retrieval
was performed, and deparaffinized tissue sections in Antigen Retrieval
Citra Solution (Biogenex Laboratories, San Ramon, CA) were treated
with microwave in a pressure cooker for 25 minutes. Tissue sections
were incubated with 3% H2O2(30 minutes) to block endogenous
peroxidase (Dako Cytomation, Carpinteria, CA). A primary antibody
[mouse monoclonal to AURKA (ab13824), 1:100 dilution; Abcam
Inc, Cambridge, MA] was applied, and the slides were maintained
at 4°C overnight, followed by mouse secondary antibody (Vector
Laboratories, Burlingame, CA) for 60 minutes, an avidin-biotin com-
plex conjugate (Vector Laboratories) for 60 minutes, diaminobenzidine
(5 minutes) and methyl-green counterstain. Nuclear AURKA expres-
sion was recorded as no expression, weak expression, moderate expres-
sion, or strong expression (Figure 1). AURKA overexpression was
defined as moderate to strong expression in any portion of tumor cells
or at least 50% of tumor cells with weak staining. Appropriate posi-
tive and negative controls were included in each run of immunohisto-
chemistry. Each immunohistochemical maker was interpreted by one of
the investigators (AURKA by Y.B.; cyclin D1 and β-catenin by K.N.;
p53, p21, COX-2, and FASN by S.O.) unaware of other data. A ran-
dom selection of 117 cases was examined for AURKA by a second
observer (K.S.) unaware of other data, and concordance between the
two observers was 0.85 (κ = 0.62, P < .0001), indicating substantial
agreement. For each of the other immunohistochemical markers, a
second observer (S.O. for β-catenin; K.S. for cyclin D1 and p21;
K.N. for p53, COX-2, and FASN) examined a random selection of
more than 100 tumors unaware of other data, and the concordance
rate between the two observers was always greater than 0.82 [all κ >
0.61 (except for FASN, κ = 0.57), all P < .0001], indicating generally
All statistical analyses used SAS program (Version 9.1; SAS Institute,
Cary, NC). All P values were two-sided, and statistical significance
was set at P ≤ .05. For categorical data, the χ2test was performed,
and odds ratio (OR) with 95% confidence interval (CI) was com-
puted. To compare mean LINE-1 methylation levels, the t test assum-
ing unequal variances was performed. The κ coefficient was calculated
to assess an agreement between the two interpreters in immunohisto-
chemical analyses. To assess independent relations of AURKA over-
expression with CIN, a multivariate logistic regression analysis was
performed. Odds ratio was adjusted for sex, age (continuous), body
mass index (BMI, <30 vs ≥30 kg/m2), family history of colorectal can-
cer (present vs absent), tumor location (right colon vs left colon vs rec-
tum), tumor stage (I-II vs III-IV), tumor grade (low vs high), mucinous
component (0 vs ≥1%), signet ring cell component (0 vs ≥1%), CIMP
status (high vs CIMP-low/0), MSI status (high vs low/MSS), LINE-1
methylation (continuous), BRAF, KRAS, PIK3CA, p53, p21, cyclin D1,
β-catenin, COX-2, and FASN.
For survival analysis, Kaplan-Meier method was used to assess sur-
vival time distribution according to AURKA status, and log-rank test
was used to test significance of a deviation from the null hypothesis.
For the analyses of colorectal cancer–specific mortality, death as a
result of colorectal cancer was the primary end point and deaths as
a result of other causes were censored. To assess independent effect of
AURKA on mortality, we constructed a multivariate, stage-matched
conditional Cox proportional hazard model to compute a hazard
ratio (HR) according to AURKA status, adjusted for sex, age, year of
diagnosis (continuous), BMI, family history of colorectal cancer, tumor
methylation, p53, p21, cyclin D1, β-catenin, COX-2, and FASN.
AURKA Overexpression in Colorectal Cancer
Among the 517 colorectal cancers in this study, 98 (19%) showed
nuclear AURKA overexpression (i.e., AURKA-positive; Figure 1).
Table 1 shows the frequencies of AURKA overexpression according to
Figure 1. AURKA (Aurora-A) expression in colorectal cancer cells.
(A) Negative for nuclear AURKA overexpression in colorectal cancer
cells (arrow). Inflammatory cells serve as an internal positive control
for AURKA positivity (white arrow). (B) Positive for nuclear AURKA
overexpression in colorectal cancer cells (arrowheads).
AURKA and CIN in Colorectal CancerBaba et al.Neoplasia Vol. 11, No. 5, 2009
various clinical and pathologicfeatures. AURKA overexpression was in-
versely associated with family history of colorectal cancer (OR, 0.49;
95% CI, 0.26-0.89; P = .018).
AURKA, CIN, and Other Molecular Features in
Table 2 summarizes the frequencies of AURKA overexpression
according to status of CIN and other molecular features in colorectal
cancer. Chromosomal instability (CIN) was defined as the presence of
LOH in any of the chromosomal segments among 2p, 5q, 17q, and
18q. AURKA overexpression was significantly associated with CIN
(OR, 2.17; 95% CI, 1.09-4.32; P = .024).
AURKA overexpression was inversely associated with KRAS muta-
tion (OR, 0.56; 95% CI, 0.35-0.92; P = .021), PIK3CA mutation
(OR, 0.25; 95% CI, 0.09-0.71; P = .0050), and β-catenin activation
(OR, 0.60; 95% CI, 0.36-0.99; P = .046; Table 2).
Multivariate Analysis to Assess Independent Relations
We performed multivariate logistic regression analysis to examine
whether AURKA was independently associated with CIN (Table 3).
AURKA overexpression was significantly associated with CIN (multi-
variate OR 2.97; 95% CI, 1.40-6.29; P = .0045) independent of other
variables. In addition, AURKA seemed to be associated with cyclin D1
Table 1. Frequency of AURKA Overexpression in Colorectal Cancer.
Clinical or Pathologic FeatureTotal NAURKA (+) Univariate OR (95% CI)P
Family history of colorectal cancer
Right colon (cecum
to transverse colon)
Left colon (splenic
flexure to sigmoid)
Signet ring cell component
243 53 (22%)1
165 25 (15%) 0.64 (0.38-1.08)
89 14 (16%)0.66 (0.35-1.28)
Only significant P values are described.
Table 2. Frequency of AURKA Overexpression in Colorectal Cancer According to
Various Molecular Features.
Molecular FeatureTotal NAURKA (+) Univariate OR (95% CI) P
CIMP status (no. of methylated CIMP markers)
Cyclin D1 expression
Inactive (score 0-2) 294
Active (score 3-5)175
1.51 (0.83-2.77) 67
61 9 (15%)
0.48 (0.21-1.10) 65
*Chromosomal instability was defined as the presence of LOH in any of the chromosomal
segments among 2p, 5q, 17q, and 18q.
†β-Catenin activation score is based on the method previously described .
Table 3. Multivariate Logistic Regression to Show Independent Relationship between
CIN and AURKA Overexpression in Colorectal Cancer.
Variable Independently Associated
Multivariate OR (95% CI)P
Other significant variables
Cyclin D1 expression
Family history of colorectal cancer
The multivariate logistic regression model included age, sex, BMI, tumor location, stage,
grade, mucinous component, signet ring cells, CIMP, MSI, KRAS, BRAF, LINE-1 methyl-
ation, p53, p21, β-catenin, COX-2, FASN,and the variables listed in the table. Only signif-
icant variables are listed.
*Chromosomal instability was defined as the presence of LOH in any of the chromo-
somal segments among 2p, 5q, 17q, and 18q.
Neoplasia Vol. 11, No. 5, 2009 AURKA and CIN in Colorectal CancerBaba et al.
expression (P = .012) and inversely with PIK3CA mutation (P = .010),
FASN expression (P = .032), and family history of colorectal cancer
(P = .036; Table 3); however, considering multiple hypotheses testing
and these P values between .05 and .01, any of these additional asso-
ciations might simply be a chance event.
AURKA and Patient Survival
We assessed the influence of AURKA overexpression on clinical
outcome of patients with stage I to IV colorectal cancer and adequate
follow-up. We have previously shown that clinical outcome data in
our two independent cohort studies are valid and reliable in detecting
significant molecular predictors of patient survival [25,26,43,44].
There were a total of 216 deaths, including 124 colorectal cancer–
specific deaths. In Kaplan-Meier analysis, AURKA was not signifi-
cantly associated with colorectal cancer–specific (log rank, P = .67)
or overall survival (log rank, P = .78). We also performed Cox regres-
sion analysis to assess patient mortality according to AURKA status
(Table 4). For both cancer-specific and overall mortality, AURKA
overexpression was not significantly related with patient outcome
in univariate analysis, stage-matched analysis, or multivariate analy-
sis. When we assessed patients with colon cancers, AURKA remained
unrelated with patient mortality.
We conducted this study to examine the relationship between
AURKA overexpression and CIN in colorectal cancer. In addition,
we assessed the relationship of AURKA with clinical, pathologic,
and other molecular features and with patient survival. We found that
AURKA was associated with CIN, independent of any of the clinical,
pathologic, and molecular variables examined. Our rich tumor data-
base allowed us to examine whether there is an independent relation-
ship of AURKA with CIN as well as with clinical outcome. Our data
support the hypothesis that AURKA overexpression is one of the con-
tributing factors for CIN during colorectal cancer development.
Studying molecular alterations is important in cancer research [45–
56]. Our resource of a large number (N = 517) of colorectal cancers
derived from the two independent prospective cohort studies has en-
abled us to precisely estimate the frequency of colorectal cancers with a
specific molecular feature (such as AURKA overexpression, MSI, etc.).
The large number of cases has also provided us with a sufficient power
in the multivariate logistic regression analysis and survival analysis.
Thus, in survival analysis, we can conclude that AURKA expression
is not significantly associated with patient survival.
Accumulating evidence suggests that AURKA activation is related to
cancer development through CIN [11–13,57]. AURKA is mainly lo-
calized at spindle poles and the mitotic spindle during mitosis, where it
regulates the function of centrosomes, spindles, and kinetochores, all
of which are required for proper mitosis progression [9,10]. AURKA
protein expression has also been related with TERT (telomerase) activ-
ity, thus possibly related with telomere function . AURKA gene
amplification [58,59] and AURKA gene overexpression  have been
reported in colorectal cancer. Another study using colon cancer cell
lines and a small number (N = 48) of human colorectal cancer tissues
has shown that high copy number of AURKA is associated with CIN
and AURKA protein overexpression . However, the discrepancy in
the frequencies of AURKA amplification and AURKA protein over-
expression has been reported in several other cancers [13,19,60]. To
the best of our knowledge, a significant relationship between AURKA
protein overexpression and CIN has not been shown using a large
number of colorectal cancer tissues. Molecular correlates with AURKA
protein overexpression are important for the better understanding of
genetic and epigenetic alterations during the colorectal carcinogenic
process, especially in relation to CIN.
It remains controversial whether AURKA expression level correlates
with malignant phenotype or patient prognosis in human cancers.
Studies on several types of human cancers have indicated that AURKA
overexpression may be related with poor prognosis [15,17,18,61] or
with higher recurrence rate . However, other studies have shown
that AURKA expression is not associated with patient prognosis
[20,63]. Another study has shown that activation of AURKA is asso-
ciated with an early stage disease in ovarian cancer . In a previous
study on colorectal cancer (N = 200) , AURKA overexpression
was associated with high-grade tumor, but the relationship between
AURKA and clinical outcome was unclear. In our current study (N =
517), AURKA overexpression was not associated with tumor grade.
This discrepancy might be due to a difference in the patient cohorts
or the methods to assess AURKA overexpression or simply due to a
chance variation between different studies. In addition, our analysis
found that AURKA overexpression was not associated with prognosis
of patients with stage I to IV colorectal cancer. Our findings suggest
that AURKA overexpression may not mark an aggressive type of colo-
Interestingly, we did observe inverse relations between AURKA
and PIK3CA mutation as well as FASN expression in colorectal can-
cer, independent of clinical, pathologic, and other molecular vari-
ables. In various cancers including colorectal cancer, mutant PIK3CA
stimulates the PI3K-AKT signaling pathway . Fatty acid synthase
overexpression has been associated with the PI3K-AKT pathway
Table 4. AURKA Expression and Patient Mortality in Colorectal Cancer.
Total N Cancer-Specific MortalityOverall Mortality
Deaths/Person-Years Univariate HR
Deaths/Person-Years Univariate HR
Colon and rectal cancers
0.90 (0.57-1.45) 0.98 (0.60-1.60)
1 (referent) 1 (referent)
0.95 (0.67-1.36) 1.00 (0.69-1.45)
1 (referent) 1 (referent)
1.07 (0.65-1.77) 1.13 (0.66-1.93)
1 (referent)1 (referent)
0.99 (0.67-1.46) 1.00 (0.66-1.50)
1 (referent)1 (referent)
The multivariate, stage-matched conditional Cox model included sex, age, year of diagnosis (continuous), BMI, family history of colorectal cancer, tumor location, stage, grade, CIMP, MSI,
CIN, BRAF, KRAS, PIK3CA, LINE-1 methylation, p53, p21, cyclin D1, β-catenin, COX-2, and FASN.
AURKA and CIN in Colorectal Cancer Baba et al.Neoplasia Vol. 11, No. 5, 2009
activation in some cancers [65,66]. A study using human cancer cell
of AURKA for mitotic progression. In contrast, another study using
mouse oocytes  has shown that the activation of AURKA on
microtubule-organizing centers is independent of the PI3K-AKT path-
way. As to the relationship between AURKA and the PI3K-AKT path-
way, further studies are needed. We also demonstrated that AURKA
overexpression was associated with nuclear cyclin D1 expression. A
previous study using a mouse model  has reported that AURKA
overexpression cause nuclear accumulation of cyclin D1, which is in
agreement with our current data.
Recently, Aurora kinases have been targeted for cancer therapy,
and a new class of drugs known as Aurora kinase inhibitors has been
undergoing preclinical and clinical assessments [69,70]. Among
them, VX-680 has shown promising results in animal studies, in-
hibiting tumor growth in a range of xenograft models and leading
to regression of colon tumor . VX-680 is already undergoing
the clinical study, but there is no biomarker for selecting patients
to benefit for clinical trials of this drug. Hereafter, AURKA expres-
sion in the resected specimens might attract increasing attention as a
biomarker for patient selection. In this respect, our findings may
have clinical implications.
In particular, a possible inverse relation between AURKA and family
history of colorectal cancer merits discussion. An association between
a molecular change and family history of colorectal cancer implies a
genetic factor (and/or a shared environmental factor) that may con-
tribute to the development of the given molecular change. The inverse
association between AURKA and family history likely supports genetic
predisposition to molecular changes (such as MSI) alternative to the
AURKA-CIN pathway. Clarification of this issue by future research
is important considering AURKA inhibitors as potential targeted ther-
apy against colorectal cancers, including familial cases.
In conclusion, using a large number of colorectal cancers, we have
shown that AURKA overexpression is independently associated with
CIN. Our data support the hypothesis that AURKA may contribute
to colorectal carcinogenesis through CIN.
The authors thank the NHS and HPFS cohort participants who have
generously agreed to provide biological specimens and information
through responses to questionnaires. The authors thank Frank Speizer,
Walter Willett, Susan Hankinson, Graham Colditz, Meir Stampfer,
and many other staff members who implemented and have maintained
the cohort studies.
 Grady WM and Carethers JM (2008). Genomic and epigenetic instability in
colorectal cancer pathogenesis. Gastroenterology 135, 1079–1099.
 Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler
KW, and Vogelstein B (1998). Mutations of mitotic checkpoint genes in human
cancers. Nature 392, 300–303.
 Gualberto A, Aldape K, Kozakiewicz K, and Tlsty TD (1998). An oncogenic
form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle
checkpoint control. Proc Natl Acad Sci USA 95, 5166–5171.
 Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B,
and Lengauer C (2004). Inactivation of hCDC4 can cause chromosomal instabil-
ity. Nature 428, 77–81.
 Maser RS and DePinho RA (2002). Connecting chromosomes, crisis, and cancer.
Science 297, 565–569.
 Fukasawa K (2007). Oncogenes and tumour suppressors take on centrosomes.
Nat Rev Cancer 7, 911–924.
 Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, and
Doxsey SJ (1998). Centrosome defects and genetic instability in malignant tumors.
Cancer Res 58, 3974–3985.
 Lingle WL, Barrett SL, Negron VC, D’Assoro AB, Boeneman K, Liu W,
Whitehead CM, Reynolds C, and Salisbury JL (2002). Centrosome amplification
drives chromosomal instability in breast tumor development. Proc Natl Acad Sci
USA 99, 1978–1983.
 Fu J, Bian M, Jiang Q, and Zhang C (2007). Roles of Aurora kinases in mitosis
and tumorigenesis. Mol Cancer Res 5, 1–10.
 Marumoto T, Zhang D, and Saya H (2005). Aurora-A—a guardian of poles.
Nat Rev Cancer 5, 42–50.
 Wang X, Zhou YX, Qiao W, Tominaga Y, Ouchi M, Ouchi T, and Deng CX
(2006). Overexpression of aurora kinase A in mouse mammary epithelium in-
duces genetic instability preceding mammary tumor formation. Oncogene 25,
 Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, Schryver B,
Flanagan P, Clairvoyant F, Ginther C, et al. (1998). A homologue of Drosophila
aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J
 Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, Brinkley BR, and Sen S
(1998). Tumour amplified kinase STK15/BTAK induces centrosome amplifica-
tion, aneuploidy and transformation. Nat Genet 20, 189–193.
 Jeng YM, Peng SY, Lin CY, and Hsu HC (2004). Overexpression and amplifica-
tion of Aurora-A in hepatocellular carcinoma. Clin Cancer Res 10, 2065–2071.
 Landen CN Jr, Lin YG, Immaneni A, Deavers MT, Merritt WM, Spannuth
WA, Bodurka DC, Gershenson DM, Brinkley WR, and Sood AK (2007). Over-
expression of the centrosomal protein Aurora-A kinase is associated with poor
prognosis in epithelial ovarian cancer patients. Clin Cancer Res 13, 4098–4104.
 Lam AK, Ong K, and Ho YH (2008). Aurora kinase expression in colorectal
adenocarcinoma: correlations with clinicopathological features, p16 expression,
and telomerase activity. Hum Pathol 39, 599–604.
 Tanaka E, Hashimoto Y, Ito T, Okumura T, Kan T, Watanabe G, Imamura M,
Inazawa J, and Shimada Y (2005). The clinical significance of Aurora-A/STK15/
BTAK expression in human esophageal squamous cell carcinoma. Clin Cancer
Res 11, 1827–1834.
 Nadler Y, Camp RL, Schwartz C, Rimm DL, Kluger HM, and Kluger Y (2008).
Expression of Aurora A (but not Aurora B) is predictive of survival in breast cancer.
Clin Cancer Res 14, 4455–4462.
SV, and Cheng JQ (2003). Activation and overexpression of centrosome kinase
BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res 9, 1420–1426.
 Kurai M, Shiozawa T, Shih HC, Miyamoto T, Feng YZ, Kashima H, Suzuki A,
and Konishi I (2005). Expression of Aurora kinases A and B in normal, hyper-
plastic, and malignant human endometrium: Aurora B as a predictor for poor
prognosis in endometrial carcinoma. Hum Pathol 36, 1281–1288.
 Nishida N, Nagasaka T, Kashiwagi K, Boland CR, and Goel A (2007). High
copy amplification of the Aurora-A gene is associated with chromosomal insta-
bility phenotype in human colorectal cancers. Cancer Biol Ther 6, 525–533.
 Colditz GA and Hankinson SE (2005). The Nurses’ Health Study: lifestyle and
health among women. Nat Rev Cancer 5, 388–396.
 Chan AT, Ogino S, and Fuchs CS (2007). Aspirin and the risk of colorectal
cancer in relation to the expression of COX-2. N Engl J Med 356, 2131–2142.
 Ogino S, Kawasaki T, Kirkner GJ, Kraft P, Loda M, and Fuchs CS (2007).
Evaluation of markers for CpG island methylator phenotype (CIMP) in colo-
rectal cancer by a large population-based sample. J Mol Diagn 9, 305–314.
 Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES,
Giovannucci EL, and Fuchs CS (2008). A cohort study of tumoral LINE-1 hypo-
methylation and prognosis in colon cancer. J Natl Cancer Inst 100, 1734–1738.
 Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci
EL, and Fuchs CS (2009). CpG island methylator phenotype, microsatellite insta-
bility, BRAF mutation and clinical outcome in colon cancer. Gut 58, 90–96.
 Ogino S, Brahmandam M, Cantor M, Namgyal C, Kawasaki T, Kirkner G,
Meyerhardt JA, Loda M, and Fuchs CS (2006). Distinct molecular features
of colorectal carcinoma with signet ring cell component and colorectal carcinoma
with mucinous component. Mod Pathol 19, 59–68.
 Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, Mino-
Kenudson M, Lauwers GY, Loda M, and Fuchs CS (2005). Sensitive sequencing
method for KRAS mutation detection by pyrosequencing. J Mol Diagn 7, 413–421.
Neoplasia Vol. 11, No. 5, 2009AURKA and CIN in Colorectal Cancer Baba et al.
 Ogino S, Kawasaki T, Kirkner GJ, Loda M, and Fuchs CS (2006). CpG island
methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations
with male sex and KRAS mutations. J Mol Diagn 8, 582–588.
 Nosho K, Kawasaki T, Ohnishi M, Suemoto Y, Kirkner GJ, Zepf D, Yan L,
Longtine JA, Fuchs CS, and Ogino S (2008). PIK3CA mutation in colorectal can-
cer: relationship with genetic and epigenetic alterations. Neoplasia 10, 534–541.
 Nosho K, Shima K, Kure S, Irahara N, Baba Y, Chen L, Kirkner GJ, Fuchs CS,
and Ogino S (2009). JC virus T-antigen in colorectal cancer is associated with p53
expression and chromosomal instability, independent of CpG island methylator
phenotype. Neoplasia 11, 87–95.
 Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D,
Makrigiorgos GM, Weisenberger DJ, Laird PW, Loda M, et al. (2006). Pre-
cision and performance characteristics of bisulfite conversion and real-time
PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn 8,
 Ogino S, Cantor M, Kawasaki T, Brahmandam M, Kirkner GJ, Weisenberger
DJ, Campan M, Laird PW, Loda M, and Fuchs CS (2006). CpG island
methylator phenotype (CIMP) of colorectal cancer is best characterised by quan-
titative DNA methylation analysis and prospective cohort studies. Gut 55,
 Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA,
Kang GH, Widschwendter M, Weener D, Buchanan D, et al. (2006). CpG
island methylator phenotype underlies sporadic microsatellite instability and
is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 38,
 Nosho K, Irahara N, Shima K, Kure S, Kirkner GJ, Schernhammer ES, Hazra A,
Hunter DJ, Quackenbush J, Spiegelman D, et al. (2008). Comprehensive bio-
statistical analysis of CpG island methylator phenotype in colorectal cancer using
a large population-based sample. PLoS ONE 3, e3698.
 Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, and Fuchs
CS (2008). LINE-1 hypomethylation is inversely associated with microsatellite
instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer
 Ogino S, Brahmandam M, Kawasaki T, Kirkner GJ, Loda M, and Fuchs CS
(2006). Combined analysis of COX-2 and p53 expressions reveals synergistic
inverse correlations with microsatellite instability and CpG island methylator
phenotype in colorectal cancer. Neoplasia 8, 458–464.
 Ogino S, Kawasaki T, Kirkner GJ, Yamaji T, Loda M, and Fuchs CS (2007).
Loss of nuclear p27 (CDKN1B/KIP1) in colorectal cancer is correlated with
microsatellite instability and CIMP. Mod Pathol 20, 15–22.
 Ogino S, Meyerhardt JA, Cantor M, Brahmandam M, Clark JW, Namgyal C,
Kawasaki T, Kinsella K, Michelini AL, Enzinger PC, et al. (2005). Molecular
alterations in tumors and response to combination chemotherapy with gefitinib
for advanced colorectal cancer. Clin Cancer Res 11, 6650–6656.
 Ogino S, Kawasaki T, Kirkner GJ, Ogawa A, Dorfman I, Loda M, and Fuchs
CS (2006). Down-regulation of p21 (CDKN1A/CIP1) is inversely associated
with microsatellite instability and CpG island methylator phenotype (CIMP)
in colorectal cancer. J Pathol 210, 147–154.
 Nosho K, Kawasaki T, Chan AT, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs
CS, and Ogino S (2008). Cyclin D1 is frequently overexpressed in microsatellite
unstable colorectal cancer, independent of CpG island methylator phenotype.
Histopathology 53, 588–598.
 Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Dehari R, Meyerhardt
JA, Fuchs CS, and Ogino S (2007). Correlation of beta-catenin localization with
cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in
colorectal cancer. Neoplasia 9, 569–577.
 Ogino S, Kirkner GJ, Nosho K, Irahara N, Kure S, Shima K, Hazra A, Chan
AT, Dehari R, Giovannucci EL, et al. (2008). Cyclooxygenase-2 expression is
an independent predictor of poor prognosis in colon cancer. Clin Cancer Res 14,
 Ogino S, Nosho K, Meyerhardt JA, Kirkner GJ, Chan AT, Kawasaki T,
expression and patient survival in colon cancer. J Clin Oncol 26, 5713–5720.
 Roschke AV, Glebov OK, Lababidi S, Gehlhaus KS, Weinstein JN, and Kirsch IR
(2008). Chromosomal instability is associated with higher expression of genes
implicated in epithelial-mesenchymal transition, cancer invasiveness, and metastasis
and with lower expression of genes involved in cell cycle checkpoints, DNA repair,
and chromatin maintenance. Neoplasia 10, 1222–1230.
 TomaMI,GrosserM, HerrA, Aust DE, MeyeA, Hoefling C, FuesselS,Wuttig D,
Wirth MP, and Baretton GB (2008). Loss of heterozygosity and copy number
abnormality in clear cell renal cell carcinoma discovered by high-density Affymetrix
10K single nucleotide polymorphism mapping array. Neoplasia 10, 634–642.
 Wang S, Liu H, Ren L, Pan Y, and Zhang Y (2008). Inhibiting colorectal carci-
noma growth and metastasis by blocking the expression of VEGF using RNA
interference. Neoplasia 10, 399–407.
 Ahlquist T, Bottillo I, Danielsen SA, Meling GI, Rognum TO, Lind GE,
Dallapiccola B, and Lothe RA (2008). RAS signaling in colorectal carcinomas
through alteration of RAS, RAF, NF1, and/or RASSF1A. Neoplasia 10, 680–686.
 Chung YL, Troy H, Kristeleit R, Aherne W, Jackson LE, Atadja P, Griffiths JR,
Judson IR, Workman P, Leach MO, et al. (2008). Noninvasive magnetic resonance
spectroscopic pharmacodynamic markers of a novel histone deacetylase inhibitor,
LAQ824, in human colon carcinoma cells and xenografts. Neoplasia 10, 303–313.
 Derks S, Postma C, Carvalho B, van den Bosch SM, Moerkerk PT, Herman JG,
Weijenberg MP, de Bruine AP, Meijer GA, and van Engeland M (2008). In-
tegrated analysis of chromosomal, microsatellite and epigenetic instability in
colorectal cancer identifies specific associations between promoter methylation
of pivotal tumour suppressor and DNA repair genes and specific chromosomal
alterations. Carcinogenesis 29, 434–439.
 Henkhaus RS, Roy UK, Cavallo-Medved D, Sloane BF, Gerner EW, and
Ignatenko NA (2008). Caveolin-1–mediated expression and secretion of kallikrein
6 in colon cancer cells. Neoplasia 10, 140–148.
 Ueno K, Hiura M, Suehiro Y, Hazama S, Hirata H, Oka M, Imai K, Dahiya R,
and Hinoda Y (2008). Frizzled-7 as a potential therapeutic target in colorectal
cancer. Neoplasia 10, 697–705.
 Xiong H, Zhang ZG, Tian XQ, Sun DF, Liang QC, Zhang YJ, Lu R, Chen YX,
and Fang JY (2008). Inhibition of JAK1, 2/STAT3 signaling induces apopto-
sis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells.
Neoplasia 10, 287–297.
 Gorringe KL, Choong DY, Williams LH, Ramakrishna M, Sridhar A, Qiu W,
Bearfoot JL, and Campbell IG (2008). Mutation and methylation analysis of the
chromodomain-helicase-DNA binding 5 gene in ovarian cancer. Neoplasia 10,
 Potter N, Karakoula A, Phipps KP, Harkness W, Hayward R, Thompson DN,
Jacques TS, Harding B, Thomas DG, Palmer RW, et al. (2008). Genomic dele-
tions correlate with underexpression of novel candidate genes at six loci in pediatric
pilocytic astrocytoma. Neoplasia 10, 757–772.
 Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs CS, and
Ogino S (2007). IGFBP3 promoter methylation in colorectal cancer: relation-
ship with microsatellite instability, CpG island methylator phenotype, and p53.
Neoplasia 9, 1091–1098.
 Meraldi P, Honda R, and Nigg EA (2002). Aurora-A overexpression reveals tetra-
ploidization as a major route to centrosome amplification in p53−/− cells. EMBO J
 Carvalho B, Postma C, Mongera S, Hopmans E, Diskin S, van de Wiel MA, van
Criekinge W, Thas O, Matthai A, Cuesta MA, et al. (2009). Multiple putative
oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma
to carcinoma progression. Gut 58, 79–89.
 Killian A, Di Fiore F, Le Pessot F, Blanchard F, Lamy A, Raux G, Flaman JM,
Paillot B, Michel P, Sabourin JC, et al. (2007). A simple method for the routine
detection of somatic quantitative genetic alterations in colorectal cancer. Gastro-
enterology 132, 645–653.
 Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M, Masuda K,
Shimomura K, Nakamura Y, Inazawa J, Abe T, et al. (2001). Tumour-amplified
kinase BTAK is amplified and overexpressed in gastric cancers with possible
involvement in aneuploid formation. Br J Cancer 84, 824–831.
 Ogawa E, Takenaka K, Katakura H, Adachi M, Otake Y, Toda Y, Kotani H,
Manabe T, Wada H, and Tanaka F (2008). Perimembrane Aurora-A expression
is a significant prognostic factor in correlation with proliferative activity in non–
small-cell lung cancer (NSCLC). Ann Surg Oncol 15, 547–554.
 Comperat E, Camparo P, Haus R, Chartier-Kastler E, Radenen B, Richard F,
Capron F, and Paradis V (2007). Aurora-A/STK-15 is a predictive factor for
recurrent behaviour in non-invasive bladder carcinoma: a study of 128 cases
of non-invasive neoplasms. Virchows Arch 450, 419–424.
 Royce ME, Xia W, Sahin AA, Katayama H, Johnston DA, Hortobagyi G, Sen S,
and Hung MC (2004). STK15/Aurora-A expression in primary breast tumors is
correlated with nuclear grade but not with prognosis. Cancer 100, 12–19.
 Samuels Yand Velculescu VE (2004). Oncogenic mutations of PIK3CA in human
cancers. Cell Cycle 3, 1221–1224.
 Van de Sande T, Roskams T, Lerut E, Joniau S, Van Poppel H, Verhoeven G,
and Swinnen JV (2005). High-level expression of fatty acid synthase in human
AURKA and CIN in Colorectal Cancer Baba et al. Neoplasia Vol. 11, No. 5, 2009
prostate cancer tissues is linked to activation and nuclear localization of Akt/ Download full-text
PKB. J Pathol 206, 214–219.
 Wang HQ, Altomare DA, Skele KL, Poulikakos PI, Kuhajda FP, Di Cristofano
A, and Testa JR (2005). Positive feedback regulation between AKT activation
and fatty acid synthase expression in ovarian carcinoma cells. Oncogene 24,
 Liu X, Shi Y, Woods KW, Hessler P, Kroeger P, Wilsbacher J, Wang J, Wang JY,
Li C, Li Q, et al. (2008). Akt inhibitor a-443654 interferes with mitotic pro-
gression by regulating aurora a kinase expression. Neoplasia 10, 828–837.
 Saskova A, Solc P, Baran V, Kubelka M, Schultz RM, and Motlik J (2008).
Aurora kinase A controls meiosis I progression in mouse oocytes. Cell Cycle 7,
 Agnese V, Bazan V, Fiorentino FP, Fanale D, Badalamenti G, Colucci G, Adamo
V, Santini D, and Russo A (2007). The role of Aurora-A inhibitors in cancer
therapy. Ann Oncol 18 (Suppl 6), vi47–vi52.
 Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN Jr, and Gandara
DR (2008). Aurora kinases as anticancer drug targets. Clin Cancer Res 14,
 Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO,
Nakayama T, Graham JA, Demur C, Hercend T, Diu-Hercend A, et al. (2004).
VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases,
suppresses tumor growth in vivo. Nat Med 10, 262–267.
 Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, Meltzer
SJ, Wright C, Searle J, Young J, et al. (1999). Characterisation of a subtype of
colorectal cancer combining features of the suppressor and mild mutator pathways.
J Clin Pathol 52, 455–460.
Neoplasia Vol. 11, No. 5, 2009 AURKA and CIN in Colorectal CancerBaba et al.