Overexpression of the Aldo-Keto Reductase Family Protein AKR1B10 Is Highly Correlated with Smokers' Non-Small Cell Lung Carcinomas
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1776 Vol. 11, 1776 – 1785, March 1, 2005 Clinical Cancer Research
Overexpression of the Aldo-Keto Reductase Family Protein
AKR1B10 Is Highly Correlated with Smokers’
Non–Small Cell Lung Carcinomas
Shin-ichi Fukumoto,1,8 Naoko Yamauchi,3 Results: Seven genes, including aldo-keto reductase
Hisashi Moriguchi,2 Yoshitaka Hippo,1 family 1, member B10 (AKR1B10), were identified as SCC-
specific genes. AKR1B10 was further examined by
Akira Watanabe,1 Junji Shibahara,3 immunohistochemical analysis of 101 non – small cell lung
Hirokazu Taniguchi,3 Shumpei Ishikawa,1 carcinomas (NSCLC) and its overexpression was observed in
Hirotaka Ito,1 Shogo Yamamoto,1 Hiroko Iwanari,5 27 of 32 (84.4%) SCCs and 19 of 65 (29.2%) adenocarcino-
Mitsugu Hironaka,6 Yuichi Ishikawa,4 mas. Multiple regression analysis showed that smoking was
Toshiro Niki,3 Yasunori Sohara,7 an independent variable responsible for AKR1B10 over-
expression in NSCLCs (P < 0.01) and adenocarcinomas
Tatsuhiko Kodama,2 Masaharu Nishimura,8
(P < 0.01). AKR1B10 staining was occasionally observed
Masashi Fukayama,3 Hirotoshi Dosaka-Akita,9 even in squamous metaplasia, a precancerous lesion of SCC.
and Hiroyuki Aburatani1 Conclusion: AKR1B10 was overexpressed in most cases
1
Genome Science Division and 2Laboratory for Systems Biology and with SCC, which is closely associated with smoking, and
Medicine, Research Center for Advanced Science and Technology, and many adenocarcinoma cases of smokers. These results
3
Department of Pathology, Graduate School of Medicine, University of
Tokyo; 4Department of Pathology, Cancer Institute, Japanese Foundation
suggest that AKR1B10 is a potential diagnostic marker
for Cancer Research and 5Perseus Proteomics, Inc., Tokyo, Japan; specific to smokers’ NSCLCs and might be involved in
Departments of 6Pathology and 7Thoracic Surgery, Jichi Medical School, tobacco-related carcinogenesis.
Tochigi, Japan; and 8First Department of Medicine and 9Department of
Medical Oncology, Hokkaido University Graduate School of Medicine,
Sapporo, Japan INTRODUCTION
Lung cancer is the leading cause of cancer death among all
types of cancers and continues to increase in frequency
ABSTRACT worldwide (1). There are two major types of lung cancer, small
Purpose: Squamous cell carcinoma (SCC) and adenocar- cell lung carcinoma (SCLC) and non – small cell lung carcinoma
cinoma of the lung are currently subject to similar treatment (NSCLC), which account for 20% and 80% of all cases (2),
regimens despite distinct differences in histology and epide- respectively. NSCLC is further classified into squamous cell
miology. The aim of this study is to identify a molecular lung carcinoma (SCC) and lung adenocarcinoma. Despite
target with diagnostic and therapeutic values for SCC. distinct differences in histologic and epidemiologic features,
Experimental Design: Genes specifically up-regulated in adenocarcinoma and SCC are similarly treated in clinical
SCC were explored through microarray analysis of 5 SCCs, practice (3) partly because underlying molecular mechanisms
5 adenocarcinomas, 10 small cell lung carcinomas, 27 normal are largely unknown. Even the most recent therapeutic
tissues, and 40 cancer cell lines. Clinical usefulness of these innovations for NSCLC have yielded little improvement to
genes was subsequently examined mainly by immunohisto- prognosis with overall 5-year survival rates stillClinical Cancer Research 1777
molecules are not involved in carcinogenesis and inappropriate other RNA from normal tissues analyzed here is provided on
as therapeutic targets. Thus, search for genes specific to SCC request or is available at http://www.lsbm.org/db/index.html.
alone will lead to identification of a novel molecular target of Microarray analysis was done essentially as described previously
SCC, which may help developing both early detection of SCC (24). For global normalization, the average signal in an array was
and personalized therapeutics of SCC. made equal to 100.
Microarray analysis has been applied to several aspects of Systematic Selection of SCC-Specific Genes Based on
cancer research, including classification, mechanistic elucida- Microarray Analysis. We systematically explored SCC-
tion, discovery of therapeutic targets, and development of tumor specific genes that were defined as follows: its expression level
makers (16 – 21). For example, we recently explored potential is (a) up-regulated in SCC but minimal in (b) normal lung and
diagnostic or therapeutic markers of hepatocellular carcinoma bronchial epithelia, (c) adenocarcinoma and SCLC, and (d)
using microarray analysis and showed that soluble glypican-3 is normal squamous epithelia, such as skin. Briefly, genes with a
a novel serologic marker essential for early detection of median signal score across 5 SCCs of >150 and >10 times that of
hepatocellular carcinoma (19). Recent reports on microarray normal lung were first selected. Among the 136 genes selected,
analysis of lung cancer have shown that SCC and adenocarci- genes with signal score of >150 in skin and small airway
noma have different gene expression signatures, suggesting epithelial cell were omitted. We subsequently eliminated genes
involvement of distinct pathways in carcinogenesis (22, 23). In with signal >150 in SCLCs, adenocarcinomas, and most other
the present study, we searched for genes specifically overex- normal tissues and various primary culture cells. Among
pressed in SCC through microarray analysis and identified seven 12 genes selected thus far, we additionally eliminated 5 genes
genes, including aldo-keto reductase family 1, member B10 that showed low expression throughout all of 40 cancer cell
(AKR1B10). We investigated potential relevance of AKR1B10 in lines, suggesting expression by surrounding stromal cells but not
NSCLCs with a newly generated monoclonal antibody and by cancer cells (Table 1).
found that it is overexpressed in smokers’ NSCLCs, including Quantitative Real-time Reverse Transcription-PCR.
most cases with SCC. After digesting genomic DNA using DNase I (Invitrogen,
Carlsbad, CA) cDNA was synthesized from 1 Ag total RNA
MATERIALS AND METHODS using SuperScript First-Strand Synthesis System for reverse
Tissue Samples and Cell Lines. Forty-five primary lung transcription-PCR (Invitrogen) in 24 AL volume and diluted up
cancers (15 SCCs, 20 adenocarcinomas, and 10 SCLCs) were to 80 AL. Quantitative real-time PCR for AKR1B10 were done
obtained with informed consent from patients who underwent using an iCycler iQ Detection System (Bio-Rad, Hercules, CA).
lobectomy at Jichi Medical School Hospital (Tochigi, Japan), Reaction mixtures contained SYBR Green I nucleic acid gel
Cancer Institute Hospital, Japanese Foundation for Cancer stain (BMA, Rockland, ME) and primers 5V-CCCAAAGATGA-
Research (Tokyo, Japan), and Hokkaido University Medical TA AA GG TAAT GCC ATC GG T- 3 V a n d 5 V-C GAT CTG -
Hospital (Hokkaido, Japan). All samples were immediately GAAGTGGCTGAAATTGGAGA-3V for AKR1B10 or 5V-
frozen after resection and stored at 80jC until RNA or protein AGAAGGAGATCACTGCCCTGGCACC-3V and 5V-CCTG-
was extracted. Adenocarcinoma cell lines A549, H23, H522, CTTGCTGATCCACATCTGCTG-3Vfor b-actin. PCR condition
H1648, and H2347 were purchased from the American Type was 1 cycle of 94jC for 3 minutes followed by 40 cycles at
Culture Collection (Manassas, VA). SCLC cell line Lu130 and 94jC for 30 seconds, 65jC for 30 seconds, and 72jC for 1
SCC cell line H157 were obtained from Cell Resource Center for minute. All the samples were run in triplicate, and the results
Biomedical Research, Tohoku University (Miyagi, Japan). were averaged. Specific amplification of AKR1B10 was
RNA Extraction and Microarray Analysis. Tissues or confirmed by the gel electrophoresis and melting curve analysis
cells were directly lysed in Isogen reagent (Nippon Gene, Osaka, after PCR. The expression level of AKR1B10 was indicated as
Japan) and homogenized. Total RNA was extracted according to a relative ratio of its signal to that of b-actin to normalize the
manufacturer’s instructions. Surgically resected lung tissues and starting amount of template cDNA. We also did semiquantitative
lung cancers, including 5 SCCs, 10 SCLCs, a pooled sample PCR using the same condition for six pair-samples of SCC and
made up of 12 adenocarcinomas, other 5 adenocarcinomas, and 1 corresponding noncancerous lung tissues.
normal lung, were analyzed on GeneChip HG U133 oligonucle- Generation of Anti-AKR1B10 Monoclonal Antibodies.
otide arrays (Affymetrix, Santa Clara, CA) containing probes for Monoclonal antibodies against AKR1B10 were generated as des-
f40,000 human genes. Further information on the source of cribed previously (25). Briefly, glutathione S-transferase – fused
Table 1 Genes with SCC-specific overexpression
Symbol Gene name UniGene no. Functional classification
AKR1B10 Aldo-keto reductase family 1, member B10 Hs.116724 Retinal reductase
ELAFIN Elafin Hs.112341 Protease inhibitor
AKR1C1 Aldo-keto reductase family 1, member C1 Hs.431175 Detoxification
SPRR3 Small proline-rich protein 3 Hs.139322 Structural component
ALDH3A1 Aldehyde dehydrogenase 3 family, member A1 Hs.575 Detoxification
NQO1 NAD(P)H dehydrogenase, quinone 1 Hs.406515 Activation of carcinogens
UGT1A9 UDP glycosyltransferase 1 family, polypeptide A9 Hs.375197 Detoxification
NOTE. Final seven genes selected through microarray analysis are listed. Representative function of each gene is summarized from the literature or
National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov).
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Research.1778 AKR1B10 Overexpression in Smoking-Related NSCLCs
Fig. 1 Expression pro-
files of AKR1B10. A
tissue-wide expression
of AKR1B10 was dis-
played with CYFRA and
SCCAg as references.
Signal denotes gene ex-
pression level obtained
from microarray analy-
sis: (a) 27 normal tis-
sues, (b) 5 fetal tissues,
(c ) 7 cultured normal
cells, (d) 5 adenocarci-
nomas, (e) 10 SCLCs,
(f) 5 SCCs, and (g) 7
lung cancer cell lines.
Filled columns, SCC.
full-length AKR1B10 produced in Escherichia coli was embedded tissue archive at the University of Tokyo. The sections
immunized to female BALB/c mice. Nine clones of monoclonal were deparaffinized in xylene, washed in ethanol, and rehydrated
hybridomas were selected by immunoblotting against recombi- in TBS. Antigen retrieval was done in 10 mmol/L citrate buffer
nant AKR1B10 transiently expressed in COS-7 cells. We (pH 6.0) at 120jC for 10 minutes following incubation with TBS
selected H4025 as a specific antibody in this study because a with 2% nonfat dried milk. Then, H4025 (50 Ag/mL) or
single band at around M r 36,000 was observed only in cytokeratin 5/6 (1:500, DAKO Ltd., Cambridge, United King-
AKR1B10-expressing cell lines as revealed by microarray dom) was applied for 1 hour followed by the secondary staining
analysis of 37 cell lines. with DAKO Envision+ reagent. All sections were counterstained
Immunoblot Analysis. Immunoblot analysis was done as with Mayer’s hematoxylin. We defined AKR1B10 positive if
described previously (25). Briefly, cells or tissues were lysed by >10% of tumor cells displayed immunoreactivity.
10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, We first examined archival samples of the University of
1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS with Tokyo to compare expression of AKR1B10 and that of keratin
protease inhibitor cocktail (Sigma, St. Louis, MO) at 4jC. 5/6 in NSCLCs, squamous epithelia of skin and esophagus,
H4025 (5 Ag/mL) or anti-b-actin antibody (0.3 Ag/mL, Sigma) alveolar epithelium, and bronchus. We have analyzed pre-
was used as primary antibodies. viously 217 primary NSCLC specimens for expression of cyclin
Immunocytochemistry and Confocal Microscopy Ana- E, Ki-67, Bcl-2, p53, retinoblastoma protein, p27, GalNAcT3,
lysis. Immunostaining of culture cells were done after fixation and GnT-V (8, 9, 11, 12). Among these, we next examined 101
in 4% paraformaldehyde and permeabilization in 0.2% Triton X- NSCLCs, which were classified into 32 SCCs, 65 adenocarci-
100 followed by incubation with 2% nonfat milk in TBS. An nomas, and 4 adenosquamous cell carcinomas according to
antibody H4025 (50 Ag/mL) was applied as a primary antibody WHO criteria (26). Clinicopathologic features are summarized
and incubated in a moist chamber at room temperature for 1 hour. in Table 3. The postsurgical pathologic tumor-node-metastasis
The secondary staining was done with FITC-labeled anti-mouse stage was determined according to the guidelines of the
IgG antibody (Sigma) as secondary antibody at room temper- American Joint Committee on Cancer (27). The Medical Ethical
ature for 1 hour. Dual-color detection by confocal laser scan Committee of Hokkaido University School of Medicine
microscopy (TCS SP2 system, Leica, Bensheim, Germany) was approved this immunohistochemical study.
done after treatment with a 0.5 Amol/L solution of the Statistical Analysis. We analyzed the statistical signifi-
mitochondrial stain MitoTracker Red CMXRos (Invitrogen) or cance of the relationship between the expression of AKR1B10
the intercalator of double-strand nucleic acid stain propidium and clinicopathologic variables by v 2 test, Yates v 2 test, or
iodide (Invitrogen). Fisher’s exact test as appropriate. We also analyzed the
Immunostaining Analysis. Immunohistochemical analy- associations between AKR1B10 expressions and the cyclin E
sis for AKR1B10 was done with the formalin-fixed, paraffin- or Ki-67 labeling index (%; refs. 8, 9) by Student’s t test.
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Research.Clinical Cancer Research 1779
Fig. 2 Overexpression of AKR1B10 in SCC. A, semiquantitative PCR using six pairs of SCC and noncancerous lung tissues. Note that AKR1B10 was
up-regulated in all paired samples. B, quantitative real-time PCR. Examined samples were 9 SCCs, 12 adenocarcinomas, 10 SCLCs, 6 lung cancer cell
lines, and 5 normal lung tissues. Note expression level of AKR1B10 was remarkably high in 6 SCCs compared with 4 adenocarcinomas. C, immunoblot
analysis of AKR1B10. A 36-kDa protein was detected in SCCs and AKR1B10-expressing cell lines H1648 and A549. N, normal lung tissues; C, cancer
tissues; CL, cell line.
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Research.1780 AKR1B10 Overexpression in Smoking-Related NSCLCs
We additionally used multiple regression analysis to extract Table 2 Expression of AKR1B10 and keratin 5/6 in NSCLCs and
factors responsible for AKR1B10 expression in NSCLCs and normal epithelia
adenocarcinomas alone. Sex, age, smoking history, differentia- AKR1B10 Keratin 5/6
tion, pT classification, pN classification, survival time, histology, NSCLCs SCC (n = 23) Positive (61%) Positive (83%)
cyclin E, Ki-67, GalNAcT3, and GnT-V were used as Adenocarcinoma Positive (33%) Negative (0%)
(n = 24)
independent variables and AKR1B10 expression as a dependent Normal Pulmonary alveoli Negative Negative
variable. Differences were considered significant at P < 0.05. epithelia (n = 3)
We simultaneously examined the correlation coefficient and the Bronchial epithelia Negative Positive
partial correlation coefficient between AKR1B10 expression and (n = 3)
Squamous epithelia
smoking or sex.
Skin (n = 3) Negative Positive
Esophagus Negative Positive
RESULTS (n = 3)
Microarray Analysis Identifies Seven Genes Specifically
Up-Regulated in SCC. We selected seven potential SCC-
specific genes (see Materials and Methods) using microarray number of samples revealed that AKR1B10 was expressed
analysis (Table 1). Tissue-wide expression profiles of these genes not only in most cases of SCC but also in a subset of
showed their high specificity compared with two widely used adenocarcinoma. To investigate relevance of AKR1B10 in
diagnostic markers of SCC, SCC antigen and CYFRA 21.1, NSCLCs, we subsequently did immunohistochemical analysis
suggesting robustness of our selection for SCC-specific genes in 101 primary NSCLCs, including 65 adenocarcinoma
(Fig. 1; Supplementary Fig. 1). Among these seven genes, (Table 3). AKR1B10 staining was observed in 27 of 32
AKR1C1, ELAFIN, NQO1, and UGT1A9 were reported previ- (84.4%) SCCs but also in 19 of 65 (29.2%) adenocarcinomas
ously as potential target genes for detection or therapy against (Table 3). In adenosquamous cell carcinomas, AKR1B10
lung cancer (28 – 31); SPRR3 is overexpressed in epidermal SCC staining was observed in 2 of 4 cases and restricted to SCC
(32); and ALDH3A1 was reported to be involved in metabolism of components of these 2 cases (data not shown). AKR1B10 was
tobacco carcinogens (33). Overexpression of AKR1B10 has not preferentially observed in cancer cells with obvious squamous
been reported previously in lung cancer; then, we investigated differentiation in SCC (Fig. 3A-E), whereas with lower
whether it represents a good molecular target of SCC. differentiation grade in adenocarcinoma (Fig. 3G). Interest-
Overexpression of AKR1B10 in SCC. To confirm array ingly, we occasionally observed AKR1B10 staining in lesions
data, we first did semiquantitative PCR. Overexpression of with metaplasia: squamous metaplasia (Fig. 3I) and transi-
AKR1B10 in SCC was observed in six pair-samples analysis tional cell metaplasia (Fig. 3J) in noncancerous areas (Fig. 3K)
(Fig. 2A). We subsequently investigated expression level of of one smoker’s SCC specimen. We seldom detected positive
AKR1B10 across lung cancer tissues by quantitative reverse staining in noncancerous portion of lung tissue, except two
transcription-PCR. Overexpression of AKR1B10 was observed cases in normal bronchial epithelia of smokers (Supplementary
in 6 of 9 (67%) SCCs but not in SCLCs or normal lung tissues Fig. 2A and B).
(Fig. 2B). Expression of AKR1B10 was also observed in 4 of 12 AKR1B10 staining was mainly observed in cytoplasm of
(33%) adenocarcinomas, although its level was not so high as in cancer cells but also in nucleus in a subset of cells (Fig. 3E
SCC (Fig. 2B). and F). Notably, two cases had apparent AKR1B10 staining
Next, we investigated expression of AKR1B10 protein by mainly in nuclei (Fig. 3M). These results were essentially
immunoblot analysis with a newly generated monoclonal anti- confirmed in confocal microscopy analysis of A549 cells.
AKR1B10 antibody, H4025. As for three pair-samples of SCC AKR1B10 was generally localized in cytoplasm, neither in
used in semiquantitative PCR above, AKR1B10 was observed nucleus nor in mitochondria in most cells. However, a subset of
only in cancerous tissues, whereas AKR1B10 was negative in cells had additional staining in nucleus in 70% confluency
3 adenocarcinomas (Fig. 2C). (Fig. 3L and M) but not in full confluency (Fig. 3N).
Comparison of AKR1B10 with Pan-Squamous Cell Correlation between AKR1B10 Overexpression and
Marker Keratin 5/6. As a SCC marker, keratin 5/6 is widely Smoking History in NSCLC and Adenocarcinoma. To
used based on its specificity to squamous cells. Unique feature of clarify the factors that correlate with AKR1B10 immunostain-
AKR1B10 as we identified in our selection is that it is not a ing, we carried out a statistical analysis that examined a variety
merely squamous cell – specific marker unlike keratin 5/6 but a of clinicopathologic variables and the expression of molecules
SCC-specific marker. To highlight the difference in ‘‘specificity’’ that we reported previously (refs. 8, 9, 11, 12; Table 3). We
of these two molecules, we compared their expression in observed positive correlations between AKR1B10 overexpres-
NSCLCs and normal tissues, including squamous epithelia of sion and SCCs (v 2 test, P < 0.0001) and smoking (v 2 test,
skin and esophagus, alveolar epithelium, and columnar epithelia P < 0.0001) in NSCLCs. AKR1B10 overexpression was
of bronchus (Table 2). Keratin 5/6 staining was observed in observed in 40 of 61 (65.6%) smokers’ NSCLCs. The
normal squamous epithelia, columnar epithelia, and 83% of correlation coefficient between AKR1B10 overexpression and
SCCs but not in adenocarcinoma. In contrast, AKR1B10 smoking was 0.47 in NSCLCs. Partial correlation coefficient
staining was observed in 64% of SCC and 30% of adenocar- was 0.41 even after removing the effect of positive correlation
cinoma but not in normal epithelia (Table 2). between AKR1B10 overexpression and male (P < 0.05). These
Distinct Localization of AKR1B10 in SCC and Adeno- results indicate the significant correlation between AKR1B10
carcinoma. As we described above, analysis in larger overexpression and smoking.
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Univariate analysis in NSCLCs also showed that
AKR1B10 was overexpressed in tumors with high pT
classification (P < 0.05). Additionally, AKR1B10-positive
cases had a higher Ki-67 expression (P < 0.001), higher cyclin
E expression (P < 0.01), lower GalNAcT3 expression
(P < 0.01), and lower GnT-V expression (P < 0.05) than
negative cases in NSCLCs. Student’s t test revealed that there
was a significant difference between AKR1B10 expression and
Ki-67 expression (P < 0.005) and cyclin E expression (P < 0.05)
in NSCLCs.
Multiple regression analysis showed that smoking
(P < 0.01), SCC (P < 0.01), and lower GalNAcT3 (P < 0.05)
were important independent variables responsible for
AKR1B10 overexpression in NSCLCs (Table 4). We subse-
quently analyzed only adenocarcinomas (n = 65) because most
SCCs were AKR1B10 positive (84.4%) and smokers (96.9%).
Interestingly, there was still a remarkable correlation (v 2 test,
P < 0.01) between AKR1B10 overexpression and smoking
in adenocarcinomas (Table 3). Moreover, it was also shown
that smoking was the only important independent variable
responsible for AKR1B10 expression in adenocarcinomas
(P < 0.01; Table 4).
DISCUSSION
Aldo-keto reductases are NAD(P)H-dependent oxidoreduc-
tases that catalyze the reduction of a variety of carbonyl
compounds (34). AKR1B10 is a member of this superfamily
and reduces aromatic and aliphatic aldehyde substrates (34).
Reportedly, AKR1B10 mRNA shows expression in adrenal
gland, small intestine, and colon, consistent with its putative
physiologic roles in steroid metabolism or detoxification of
reactive aldehydes in the digested food in intestinal tract (34–36).
Initial goal of our study was to identify SCC-specific
molecules, distinct from currently used SCC markers that are
specific to squamous cell in general. We eliminated these
squamous cell marker genes through our selection and
identified AKR1B10 as a gene highly specific to SCC but
not to squamous cells in general. AKR1B10 was expressed in
as many as 90% of SCC of the lung but not in normal
bronchial epithelium and squamous epithelium from skin and
esophagus. This unique feature of AKR1B10 is highlighted
when we compared the results of immunohistochemical
analysis using AKR1B10 and keratin 5/6 (Table 2). AKR1B10
was highly specific to SCC when SCC and normal epithelia
were analyzed by immunohistochemistry, although its specific-
ity and sensitivity for SCC among NSCLCs were lower than
those of keratin 5/6.
In the present study, we showed that AKR1B10 is
overexpressed in SCC, which is closely associated with Fig. 3 Immunohistochemical analysis of AKR1B10. A-E, two
representative cases in SCC. H&E staining, (A) 20 and (C) 100.
smoking. Additionally, we found AKR1B10 expression even Cancerous regions with obvious (red line) and no (blue line)
in metaplasia, which is also associated with smoking and squamous differentiation. Corresponding staining of the same sample
regarded as precancerous lesions of SCC (37, 38). Unexpect- (B and D) and another sample (E) by H4025. Note that AKR1B10 is
edly, nearly one third of the cases of adenocarcinomas stained in regions with squamous differentiation. F and G, two
representative cases in adenocarcinoma. Homogenous staining was
expressed AKR1B10, but it was revealed by multiple observed in some cases (F), whereas preferential staining in regions
regression analysis that smoking was the most important with lower differentiation was observed in most cases (G). H, typical
determinant of AKR1B10 expression in adenocarcinomas. case with nuclear staining in SCC (100). I-K, AKR1B10 staining in
metaplasia of a smoker. I, squamous metaplasia: (left) 20 and (right)
Adenocarcinomas can be clustered into several subclasses 100. J, transitional cell metaplasia: (left) 20 and (right) 100.
based on reported expression profiling (22, 23). Together with Note that these metaplastic regions are observed successively in
recent reports that f40% of adenocarcinomas occur in noncancerous regions of a case with SCC (K).
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Research.1782 AKR1B10 Overexpression in Smoking-Related NSCLCs
Fig. 3 Continued L-N, subcel-
lular localization of endogenous
AKR1B10 in A549 cells in
70% (L and M) and 100% (N)
confluency. Left, AKR1B10;
middle, MitoTracker (L ) or
propidium iodide (M and N);
right, merged image. Note nu-
clear staining (L and M) has
disappeared in 100% conflu-
ency (N).
smokers (39), there is a possibility that AKR1B10 could suppressed by retinoic acid (43). Through the analysis of many
characterize a subset of adenocarcinoma associated with cancer samples, we noticed positive staining of AKR1B10
smoking. Based on our results, AKR1B10 immunostaining even in some cases with metaplasia, precancerous lesion of
could be applied to the early detection of cancer cells or SCC. Because the number of samples that contained
atypical cells in sputum, especially in heavy smokers. metaplasia was small in the present study, this result was
Then, what could be potential roles of AKR1B10 in further investigated by another study focusing on idiopathic
multistep carcinogenesis of SCCs? There are two possibilities pulmonary fibrosis, which showed that squamous metaplasia
as follows: one is that AKR1B10 may be related to cell was positive for AKR1B10 in 23 cases of 56 squamous
proliferation. There was a positive correlation between metaplasia lesions.10 These results strongly suggest that
AKR1B10 expression and putative poor prognosis factors, AKR1B10 expression is positive in precancerous lesions and
such as high Ki-67, high cyclin E, low GalNAcT3, and low may down-regulate retinoic acid, which could lead to
GnT-V in NSCLCs (8, 9, 11, 12). Moreover, AKR1B10 was carcinogenesis of SCC. Considering that AKR1B10 is an
localized in nucleus in a fraction of cancer cells in enzyme related to detoxification and that some smokers’
subconfluent culture conditions, which disappeared under bronchial epithelia without metaplasia were positive for
confluent culture, suggesting that AKR1B10 translocates AKR1B10 staining, AKR1B10 may be directly induced by
during cell cycle and is involved in the regulation of cell some chemical compounds in tobacco, which should be
cycle in a fashion yet identified. further investigated. Interestingly, we also observed frequent
Another possibility is that AKR1B10 promotes carcino- overexpression of AKR1B10 in SCC of the laryngopharynx
genesis of SCC through its enzymatic activity that counteracts and esophagus that is closely associated with smoking
the conversion of h-carotene to retinoic acid (40). Retinoic and occasional overexpression of esophageal dysplasia and
acid induces potent differentiation and growth-suppressive
effects in diverse premalignant and malignant cells (41). In
lung, deficiencies of retinoids are reported to cause hyperplasia
10
and squamous metaplasia of airway epithelium (42) that can be Fukayama et al., in preparation.
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Research.Clinical Cancer Research 1783
hyperplasia.10 Remarkably high frequency of its up-regulation underlying these adverse effects is currently unknown, but up-
specific to SCC warrants further investigation of AKR1B10 in regulation of AKR1B10 in precancerous lesions in the
carcinogenesis of SCC. bronchial epithelium of smokers may partly explain ineffec-
Various retinoids, including h-carotene, have been shown tiveness of h-carotene observed in the lung.
previously effective for the treatment and prevention of several AKR1B10 was also overexpressed in adenocarcinoma of
cancers, including carcinoma of the breast, skin, and kidney smokers. Its staining was observed in undifferentiated region in
(44 – 49). However, clinical chemoprevention trials of lung contrast to SCC with staining in differentiated region. Together
cancer by h-carotene have failed to show its effectiveness. with its overexpression in hepatocellular carcinomas (34, 36),
Moreover, administration of h-carotene unexpectedly promoted AKR1B10 may be related to another carcinogenic pathway
tumorigenesis in smokers (50, 51). Molecular mechanism distinct from that of SCC.
Table 3 Clinicopathologic features correlated to AKR1B10 overexpression
AKR1B10 AKR1B10
Characteristics NSCLC Positive Negative P, m2 test Adenocarcinoma Positive Negative P, m2 test or Fisher test
Age (y)
Median (range) 63 (31-85)1784 AKR1B10 Overexpression in Smoking-Related NSCLCs
Table 4 Multiple regression analysis for AKR1B10
NSCLCs Adenocarcinomas
Characteristics Regression coefficient P 95% Confidence interval Regression coefficient P 95% Confidence interval
Age 0 0.432 0.15 to 0.006 0.01 0.34 0.02 to 0.007
Sex 0.19 0.191 0.474 to 0.096 0.23 0.13 0.534 to 0.071
Histology 0.31 0.004 0.518 to 0.1
Differentiation 0.05 0.694 0.286 to 0.191 0.01 0.959 0.285 to 0.271
Smoking 0.425 0.004 0.143 to 0.707 0.387 0.01 0.0962 to 0.677
pT classification 0.02 0.763 0.167 to 0.123 0.15 0.095 0.322 to 0.026
pN classification 0.022 0.727 0.101 to 0.144 0.145 0.088 0.022 to 0.313
Ki-67 2E04 0.929 0.004 to 0.004 0.01 0.077 0.011 to 6E04
Cyclin E 0 0.652 0.004 to 0.003 0.002 0.424 0.003 to 0.006
GalNAcT3 0.23 0.032 0.483 to 0.02 0.111 0.507 0.423 to 0.212
GnT-V 0.03 0.483 0.111 to 0.053 0.007 0.889 0.095 to 0.11
Survival time 2E06 0.948 7E05 to 8E05 4E05 0.383 6E05 to 1E04
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Research.Overexpression of the Aldo-Keto Reductase Family Protein
AKR1B10 Is Highly Correlated with Smokers' Non−Small Cell
Lung Carcinomas
Shin-ichi Fukumoto, Naoko Yamauchi, Hisashi Moriguchi, et al.
Clin Cancer Res 2005;11:1776-1785.
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