Med Oncol (2015) 32:373

DOI 10.1007/s12032-014-0373-1

ORIGINAL PAPER

Elevated expression of IMPDH2 is associated with progression

of kidney and bladder cancer
Jun Zou • Zhaodong Han • Liang Zhou • Chao Cai •
Hongwei Luo • Yaqiang Huang • Yuxiang Liang • Huichan He •

Funeng Jiang • Cong Wang • Weide Zhong

Received: 12 November 2014 / Accepted: 14 November 2014 / Published online: 3 December 2014
Ó Springer Science+Business Media New York 2014

Abstract Novel molecular markers for cancer progres- expression of IMPDH2 was not associated with the disease
sion are valuable for the diagnosis and evaluation of clinical stages and pathological features. The findings
treatment efficacies of the diseases. Expression of inosine suggest that overexpressed IMPDH2 can be used as a
50 -monophosphate dehydrogenase type II (IMPDH2), a biomarker for kidney and bladder cancer diagnosis and is a
rate-limiting enzyme in the de novo guanine nucleotide potential therapeutic target for the diseases.
biosynthesis, is up-regulated in various neoplasms,
including prostate cancer and patient serum. However, Keywords Inosine 50 -monophosphate dehydrogenase
whether IMPDH2 can serve as a biomarker for other uro- type II  Kidney cancer  Bladder cancer  Testis cancer
logic cancers is unknown. Paired patient tissue macroarrays
were analyzed by immunohistochemistry, the IMPDH2
protein expression in these tissues was quantitated and Introduction
expressed as immunoreactivity scores. Compared with
non-cancerous tissues, IMPDH2 protein expression levels Urologic cancers include cancers derived from bladder,
were significantly upregulated in kidney and bladder can- kidney, prostate, and testicles, all of which are relatively
cer, but no difference in testis cancer. In addition, common cancer in human. Prostate cancer, as the most
common cancer in American men, has been intensively
studied. Other urologic cancers are also receiving more
Jun Zou, Zhaodong Han and Liang Zhou have contributed equally to attentions nowadays due to their increased incidence rates.
this work. Through the last decade, several urine molecular markers for
bladder cancer (BC) diagnosis have been developed. In
Electronic supplementary material The online version of this particular, cell cycle-regulating and pro-apoptotic mole-
article (doi:10.1007/s12032-014-0373-1) contains supplementary
material, which is available to authorized users. cules, as well as epigenetic modifications, such as DNA

J. Zou  C. Cai  H. Luo  Y. Huang  W. Zhong C. Wang (&)

Guangdong Provincial Institute of Nephrology, Southern School of Pharmacy, Wenzhou Medical University,
Medical University, Guangzhou 510515, China Wenzhou 325035, Zhejiang, China
e-mail: [email protected]
J. Zou  Z. Han  L. Zhou  Y. Liang  H. He  F. Jiang 
W. Zhong (&) W. Zhong
Department of Urology, Guangdong Key Laboratory of Clinical Urology Key Laboratory of Guangdong Province, The First
Molecular Medicine and Diagnostics, Guangzhou First People’s Affiliated Hospital of Guangzhou Medical University,
Hospital, Guangzhou Medical University, Guangzhou 510180, Guangzhou Medical University, Guangzhou 510230, China
China
e-mail: [email protected] W. Zhong
Department of Urology, Huadu District People’s Hospital,
J. Zou Southern Medical University, Guangzhou 510800, China
The Third Affiliated Hospital of Guangzhou Medical University,
Guangzhou 510150, China

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methylations, have been suggested for the diagnosis and Guangzhou Medical University, P.R. China. All of the
prognosis of patients with BC [1–3]. Renal cancer includes a tissue microarrays (TMAs) were obtained from Ailinabio
diverse spectrum of tumors. Among them, renal cell carci- Co., Xi’An, China. The product number of the kidney
noma (RCC) is the most common solid tumor in the adult tissue microarray is KD244, which contains 12 primary
kidney, accounting for 87 % of all renal malignancies. In the RCC and 12 adjacent non-cancerous kidney tissues col-
past few decades, increasing numbers of RCCs have been lected from three female and nine male patients with the
diagnosed owing to improved imaging system. However, age ranging from 33 to 76 (the average age is 55). The
there is still no good biomarker for RCC diagnosis. product number of the bladder tissue microarray is
Inosine 50 -monophosphate dehydrogenase type II (IMPDH2) BL481, which contains 39 primary transitional cell car-
is the rate-limiting enzyme in the de novo guanine nucleotide cinomas and eight normal bladder tissues collected from
biosynthesis, which is required for maintaining the cellular 12 females and 35 male patients with the age ranging
guanine deoxy- and ribonucleotide pools needed for DNA and from 21 to 88 (the average age is 54). The series number
RNA synthesis. We previously reported that the increased serum of the testis microarray is TE481, which contains 36
level of IMPDH2 in PCa patients is associated with the clini- seminoma, four diffuse large B cell lymphomas, and eight
copathological features of tumor, suggesting its potential as a normal testis tissues, the patient age ranges from 15 to 81
serological tumor marker for PCa. Elevated expression of IM- (the average age is 43).
PDH2 can promote the tumor metastasis and the advanced tumor
progression of PCa [4, 5]. In mammals, there are two ubiqui-
Immunohistochemistry analysis
tously expressed IMPDH isoforms, termed IMPDH1 and IM-
PDH2, which are encoded by distinct genes with 85 % identity
The TMA slides were deparaffinized with xylene and
in their amino acid sequence [6, 7]. IMPDH1 is constitutively
then serially rehydrated with ethanol. Following a brief
expressed in normal cells. However, expression of IMPDH2 is
proteolytic digestion and a peroxidase blocking, the
increased in cells actively engaged in proliferation, including
slides were incubated with the primary antibody against
cancer cells [7]. Therefore, it has been proposed to use IMPDH2
IMPDH2 (1:200, Abcam, USA) at 4 °C overnight. After
as a biomarker for cancer diagnosis.
washing to remove unbounded primary antibodies, the
Accumulating evidence demonstrates elevated expres-
slides were incubated with the peroxidase conjugated
sion of IMPDH2 in different cancer types. It has been
secondary antibody. The specifically bound secondary
considered to use IMPDH2 as a target for chemotherapy.
antibody was detected with DAKO EnVision detection
IMPDH2 is overexpressed in methotrexate (MTX)-resis-
System (Dako Diagnostics, Switzerland). Negative
tant erythroleukemia K562 cells, treating the cells with
controls were carried out by omitting the primary
the IMPDH2 inhibitor, mycophenolic acid, increases the
antibody.
sensitivity to MTX of the cells [8]. Elevated IMPDH2
After counterstained with hematoxylin, immunostain-
expression is found in colorectal cancer (CRC) and has
ing was scored by two independent experienced patholo-
been suggested that IMPDH2 can be a biomarker and
gists, who were blinded to the clinical data and outcomes
therapeutic target for CRC [9]. It is reported that IM-
of the patients. The scores of the two pathologists were
PDH2 is a promising candidate for the stratification of
compared, and any discrepant scores were re-examined by
osteosarcoma patients into low- and high-risk groups [10].
both pathologists to achieve a consensus score. The
In addition, IMPDH2 is responsible for oxymatrine-
number of positive-stained cells in ten representative
induced Hela cell mitochondrial-related apoptosis and
microscopic fields was counted, and the percentage of
inhibition of IMPDH2 sensitizes resistance to cisplatin
positive cells was calculated. The semi-quantitative ana-
treatment of the cells [11]. As kidney cancer, bladder
lysis of the stained sections was done by light-microscopy
cancer, and testis cancer are frequent diagnosed urologic
according to the immunoreactive score (IRS) by Remmele
malignancies, it is necessary to identify the value of
and Stegner.
IMPDH2 as tumor markers for diagnosis of these cancer
types.
Statistical analysis

Materials and methods Statistical analysis was performed with the software of
SPSS 18.0 for Windows (SPSS Inc, IL, USA). Continuous
Patients and tissue samples variables were expressed as mean ± SD. The t test was
used to analyze whether the differences were statistically
This study was approved by the Ethics Committee of significant in the tissues. P values smaller than 0.05 is
Guangzhou First Municipal People’s Hospital, Affiliated considered statistically significant.

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Results Table 1 Association of IMPDH2 expression with characteristics of

kidney cancer patients
IMPDH2 expression in kidney cancer is increased Clinic data n IRS scores P value
compared with adjacent non-cancerous tissues
Tissue
IMPDH2 protein expression in kidney cancer and adjacent Cancer 12 4.67 ± 1.07 0.02
non-cancerous tissues were assessed by immunohisto- Benign 12 3.42 ± 0.99
chemistry. In cancer tissues, diffuse and moderate strong Age
positive staining was observed in the cytoplasm and C60 4 4.50 ± 1.29 0.72
membrane. In contrast, cytoplasmic staining of IMPDH2 in \60 8 4.75 ± 1.04
most non-cancerous tissues was generally weak compared Clinical stage
with the cancerous tissues (Fig. 1c). The representative BT2 6 5.17 ± 1.29 0.11
pictures of IMPDH2 staining in cancer and non-cancerous CT3 6 4.17 ± 1.04
tissues with a high magnification power are shown in Pathological stage
Fig. 1d. The average score in kidney cancer is 4.67 ± 1.07, \2 6 4.17 ± 0.75 0.25
and the adjacent non-cancerous tissues are 3.42 ± 0.99. C2 4 5.00 ± 1.41
Statistical analyses revealed that the difference is statisti-
cally significant (P \ 0.05). However, due to relatively
small sample numbers, no significant association between BC tissue array. Among the 39 cancer tissues in the tissue
the staining intensity and age, clinical stage, and patho- array, 36 tissues were IMPDH2 positive. In contrast, no
logical stage was found (P [ 0.05, Table 1). positive staining of IMPDH2 was detected in all eight non-
cancerous bladder tissues (Fig. 2a). The representative
Overexpression of IMPDH2 in bladder cancer pictures of IMPDH2 staining in BC and non-cancer tissues
are shown in Fig. 2c, d. The average score in BC is
To determine whether IMPDH2 is overexpressed in blad- 4.33 ± 1.88, while that in the normal tissue is 2.88 ± 0.35,
der cancer, the immunostaining was also performed on the (P \ 0.05), indicating that the difference is statistically

Fig. 1 Immunohistochemical staining for IMPDH2 in kidney cancer cancerous tissues (IRS: Ca = 4.67 ± 1.07 vs Benign = 3.42 ± 0.99,
and adjacent non-cancerous tissues (original magnifications 9200 and P \ 0.05). *P \ 0.05. c IMPDH2 expression was weakly or nega-
9400). a Staining pattern of the kidney TMA section. Columns 1, 3, 5 tively in the cytoplasm and on the cell membrane of adjacent non-
are cancer tissues, and columns 2, 4, 6 are adjacent non-cancerous cancerous kidney tissues. This image is enlarged picture of C4 tissue.
tissues. b IRS analysis showed immunoreactivity score in cancerous d Strong IMPDH2 staining in the cytoplasm and on the cell
tissues was significantly upregulated than that in adjacent non- membrane of tumor cells. The image is enlarged picture of D1 tissue

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Fig. 2 IMPDH2 expression in

bladder cancer and adjacent
normal tissues (original
magnifications 9200 and
9400). a Staining pattern of the
bladder TMA section. Lines A–
E are bladder cancer tissues.
Line F is benign bladder tissues.
b IRS analysis showed
immunoreactivity score in
bladder tissues was higher than
that in adjacent normal tissues
(IRS: Ca = 4.33 ± 1.88 vs
Benign = 2.88 ± 0.35,
P \ 0.05). *P \ 0.05.
c Enlarged picture of F7 tissue
shows that IMPDH2 was only
weakly expressed in adjacent
normal tissues. d Enlarged view
of B3 tissue shows that
IMPDH2 is highly abundant in
the cytoplasm and on the cell
membrane of tumor cells

Table 2 Association of IMPDH2 expression with characteristics of Table 3 Association of IMPDH2 expression with characteristics of
bladder cancer patients testis cancer patients
Clinic data n IRS scores P value Clinic data n IRS scores P value

Tissue Tissue
Cancer 39 4.33 ± 1.88 0.00 Cancer 40 4.22 ± 1.31 0.96
Benign 8 2.88 ± 0.35 Benign 8 4.25 ± 0.46
Age Age
C60 16 4.94 ± 1.29 0.02 C60 4 3.75 ± 1.50 0.45
\60 23 3.91 ± 2.13 \60 36 4.28 ± 1.30
Clinical stage Clinical stage
BT2 23 4.61 ± 1.85 0.28 BT2 23 4.61 ± 1.85 0.28
CT3 16 3.94 ± 1.91 CT3 5 4.60 ± 1.14
Pathological stage
\2 11 5.18 ± 1.60 0.08
C2 22 3.91 ± 2.05
difference was found in IMPDH2 expression between can-
cerous and normal in testis tissues (P [ 0.05, Table 3). The
representative pictures of immunohistochemistry staining of
significant (Table 2). In addition, the expression of IM- IMPDH2 protein in testis tissue are shown in Fig. 3.
PDH2 in men older than 60 years of age is higher than that
in men younger than 60 years of age (P \ 0.05). No sig-
nificantly correlations between IMPDH2 expression levels Discussion
with the clinical stage and pathological stage (P [ 0.05,
Table 2). IMPDH is a key enzyme for the synthesis of purine
nucleotides, which includes IMPDH1 and IMPDH2 [12].
No significant difference in IMPDH2 expression Increased IMPDH expression and activity has been shown
between cancerous and non-cancerous testis tissues in various tumor tissues and cells [13, 14]. Since IMPDH1
is constitutively expressed in cells and IMPDH2 is upreg-
Unlike normal bladder and kidneys, normal testis highly ulated in rapidly proliferating cells, the elevated activity
expressed IMPDH2 (4.25 ± 0.46), which was similar to the and expression of IMPDH in tumor cells is primarily
IMPDH2 expression in testis cancer (4.22 ± 1.31). No caused by upregulation of the IMPDH2 isoform [15, 16].

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Fig. 3 No difference in
IMPDH2 expression between
normal and cancer testis tissues
(original magnifications 9200
and 9400). a Staining pattern of
the testis TMA section. Lines A–
E are testis cancer tissues, and
line F is benign tissues. b No
significant difference in
IMPDH2 expression between
cancer and control groups
(P [ 0.05) (IRS:
Ca = 4.22 ± 1.31 vs
Benign = 4.25 ± 0.46,
P [ 0.05). c Enlarged image of
F5 tissue shows that IMPDH2 is
strangely expressed in the
cytoplasm and on the cell
membrane of normal testis
tissue which picked. d Enlarged
image of C8 tissue shows
diffused expression of IMPDH2
in the testis tumor tissue

Our previous study demonstrates that IMPDH2 is In conclusion, our data suggest that upregulation of IM-
overexpressed at the mRNA and proteins levels in PCa and PDH2 closely correlates with human BC and human kidney
that overexpression of IMPDH2 is a contributory factor for cancer. It is a potential biomarker for diagnosis of BC and
metastasis and progression of PCa [4]. Herein, we further kidney cancer. The association between the expression level
reported that IMPDH2 is also overexpressed at the protein of IMPDH2 and pathological features of kidney and BC was
level in bladder and kidney cancer, but not in testis cancer. inconclusive due to relatively small sample numbers. How-
The results are in line with previous report that urine IM- ever, preliminary data did show IMPDH2 expression
PDH2 in patient’s urine and plasma is associated with PCa inclines to increase in advanced diseases. Therefore, further
progression, which can be used for the prognosis of human investigation with a large sample pool is warranted.
patients with PCa [17], early stage CRC [18], hematic
tumors including myelogenous leukemia [19], and mela- Acknowledgments This work was supported by grants from
National Natural Science Foundation of China (81170699, 81272813,
noma [20]. In addition, high expression of IMPDH2 in 81200550, 81470983, 81101712), Science and Technology Project of
human CRC is negatively associated with the survival time Guangdong Province (2013B021800055), Guangzhou Municipal
of the patients. A potent IMPDH2 inhibitor, mycophenolic Science and Technology Key Project (2014J4100072), Projects of
adenosine, has been show to inhibit growth of leukemia Guangdong Key Laboratory of Clinical Molecular Medicine and
Diagnostics.
cell line K562 [21]. Together, it has been suggested that
IMPDH2 not only can serve as a biomarker for leukemia Conflict of interest The authors declare that they have no conflict
diagnosis, but also can be a potential target for treating of interests.
leukemia.
Unlike other normal tissues, normal testis highly
expressed IMPDH2 (Fig. 3). It is likely due to high cell
proliferation activity in the testis since high IMPDH References
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