Expression of glucose transporter protein 1 and p63 inserous ovarian tumor

Yu Cai1, Jian-jun Zhai1, Bi-bo Feng1, Xian-zhi Duan1 and Xiao-jin He2

Departments of 1Gynecology and Obstetrics and 2Pathology, Beijing TongRen Hospital, Capital Medical University, Beijing,China

Abstract

Aim: It has been shown that glycolytic metabolism is increased in malignant cells. Cancer cell growth is anenergy-related process supported by an increased glucose metabolism. In addition, p63, a known homolog ofp53, is expressed predominantly in basal cell and squamous cell carcinomas. The purpose of this study was toevaluate the expression of glucose transporter protein 1 (GLUT1) and p63 in patients with serous ovarian tumor(benign, borderline and malignant) and study their close relationship with the malignant transformation ofserous ovarian tumors.Methods: Two hundred formalin-fixed, paraffin-embedded sections were immunostained with rabbit anti-GLUT1 polyclonal antibody and mouse anti-p63 monoclonal antibody using the streptavidin-biotin method.The samples were as follows: 40 normal ovarian tissues, 40 serous cystadenomas, 40 borderline serouscystadenomas and 80 serous cystadenocarcinomas were stained.Result: Normal ovarian tissues showed completely negative staining for GLUT1 and p63. However, frombenign serious cystadenomas, borderline cystadenomas to cystadenocarcinomas, the expression of GLUT1and p63 grew stronger (P < 0.05). Moreover, the intensity staining of GLUT1 maintained a significant associa-tion with the expression of p63 (P < 0.05). In χ2-test analysis, expression of borderline cystadenomas andcystadenocarcinomas, intraperitoneal implants, ascites, lymph node status and International Federation ofGynecology and Obstetrics (FIGO) stage and GLUT1 expression levels have an appalling significance(P < 0.05), while FIGO stage, intraperitoneal implants and lymph node status except patient age and asciteshave a statistical significance with the expression of p63 levels (P < 0.05).Conclusion: Our findings show a progressive increase in the expression of GLUT1 and p63 from the benignserous cystadenomas, borderline cystadenomas to cystadenocarcinomas. Overexpression of GLUT1 and p63are associated with the histology FIGO stage and metastasis of the tumors. These data suggested that theexpression of GLUT1 and p63 may be closely related to the malignant transformation of serous ovarian tumors.However, the relative importance of GLUT1 and p63 in ovarian serous tumor development and tumorigenesisremains mostly unclear and awaits further investigation.Key words: glucose transporter protein 1, immunohistochemistry, p63, serous ovarian neoplasms.

Introduction

The early notion that cancer is caused by gene muta-tions controlling cell growth implied that genome

stability is important for preventing oncogenesis.Cancer cell growth is an energy-related process sup-ported by an increased glucose metabolism. Thisphenomenon suggests a need for a corresponding

Received: August 28 2013.Accepted: March 5 2014.Reprint request to: Professor Yu Cai, Department of Gynecology and Obstetrics, Beijing TongRen Hospital, Capital MedicalUniversity, Beijing Economic and Technological Development Zone no. 2 West Ring Road, Beijing 100176, China.Email: caiyu892002@sina.com

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doi:10.1111/jog.12447 J. Obstet. Gynaecol. Res. Vol. 40, No. 7: 1925–1930, July 2014

© 2014 The Authors 1925Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology

increased uptake of glucose transporter proteins.Glucose transporter protein 1 (GLUT1), a facilitativecell surface glucose transport protein, is physiologi-cally in red blood cell membranes, brain capillaryendothelium (the blood–brain barrier) and the peri-neurium of the peripheral nerve (all interfaces withcells or tissues with glucose-driven metabolism). Inaddition, expression of GLUT1 has been shown in pla-centa, basal cells of benign squamous epithelium andreactive epidermal cells.1,2

A number of immunohistochemical studies havedemonstrated aberrant expression of GLUT1 in epithe-lial neoplasms, as well as a correlation of GLUT1expression with neoplastic progression. For example,in studies of the colon, benign colonic epithelium andadenomas with tubular architecture were non-staining,whereas villous adenomas associated with a high riskof transformation into carcinoma were focally positiveat the tips of the villous fronds neoplasms.3 Recently,high GLUT1 expression has been shown to be corre-lated with a greater frequency of lymph node metasta-ses and a significant increase in mortality in patientswith colon carcinoma.4

Recently, one important signal transduction path-way component being exploited as a new tumormarker is p63. p63 is a p53 homolog that has beenshown to be useful in lung cancer diagnosis and otherdiagnostic applications.5 As is known, p53 is the mostfrequently mutated tumor suppressor gene identifiedin human cancer. Tumor suppression functions of p53stem, in part, from its capabilities to induce cell cyclearrest in late G1 and/or apoptosis in response togenotoxic stress and hypoxia. The mutational inac-tivation of p53 is associated with an increased riskof tumorigenesis.6 In contrast to p53, however, p63mutations are rarely seen in tumors. There havebeen several reports that p63 plays a regulatory rolein the normal differentiation of cells, whereas its rolein tumor biology must be elucidated.7 Recently,studies revealed that p63 is highly expressed in pro-liferating basal cells of epithelial layers, including epi-dermis, cervix and prostate, and the major p63isoforms in the basal cells lack the transactivatingdomain.8

However, hitherto a regulation of p63 isoformexpression in serous ovarian cystadenocarcinomas stillhas not been covered. Very few studies have analyzedGLUT1 expression in ovarian carcinoma tissues,1,9 andno studies have been performed for serous ovariantumor. The aims of the current study were to investi-gate the expression of GLUT1 and p63 in various types

of serous ovarian tumors and to study the effects ofGLUT1 and p63 on the development and biologicalfunctions of the ovarian tumors and their relationship,and to provide an important theoretical foundation forclinical differentiation between borderline and malig-nant ovarian tumors using genetic testing and theirtreatment by gene therapy.

Methods

Two hundred archival, routinely processed, formalin-fixed, paraffin-embedded surgical pathology speci-mens were examined. These included 40 normalovarian tissues, 40 serous cystadenomas, 40 borderlineserous cystadenomas and 80 serous cystadenocarcino-mas. GLUT1 transporters and p63 were detected byimmunohistochemistry using the labeled streptavidin-biotin procedure.10–12 Sections were deparaffinized inxylene, and cells were rehydrated in decreasingethanol solution. Endogenous peroxidase was neutral-ized with 6% hydrogen peroxide for 3 min. Theslides were subjected to heat-induced antigen retrievalusing a steamer. Then, they were washed and incu-bated for 30 min at 22°C with a GLUT1 kit (RAB0526;Maixin-Bio) and p63 kit (MS-1081; Maixin-Bio). Thesamples were then washed off and biotinylated sec-ondary antibodies (Maixin-Bio) were applied to theslides for 25 min in a humidity chamber. The slideswere again washed and incubated with streptavidinperoxidase for an additional 25 min, and then sub-merged into a 3,3′-diaminobenzidine-tetrachloridebath for 5 min. Tissues were counterstained withhematoxylin. All slides were examined by lightmicroscopy.

Two pathologists independently reviewed the slidesand were in agreement regarding the extent of immu-nohistochemical staining. Staining for GLUT1 wasobserved in the cell membrane, in yellowish brown;whereas p63 staining was predominately localized tothe cell cytoplasm, partly localized to the cell nucleus,in yellowish brown. All sections were analyzed. Inten-sity of staining was quantified with regards to the per-centage of position marking at high magnification: nomarking (–); less than 20% (1+); 20–50% (2+); and morethan 50% (3+).10–12

Statistical analysis of immunohistochemical data wasperformed using the SPSS statistical package (version17.0). Differences between groups were evaluatedusing the χ2-test and Spearman’s rank correlationcoefficient.

Y. Cai et al.

1926 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology

ResultsDifferences and correlations between theexpression of GLUT1 and p63 in different seroustumor groups

The staining of GLUT1 and p63 were completely nega-tive in 40 normal ovarian tissues. Sixty-six percent(n = 27) of the 40 borderline serous cystadenomas wereweakly positive for GLUT1, however, GLUT1 expres-sion in 40 borderline serous cystadenomas and 80serous cystadenocarcinomas was 100% positive, but itis clear that strong staining (3+) was present in 81%(n = 65) of the malignancies versus 0% in the borderlinegroup (Fig. 1; see Table 1).

p63 was noted in 28 patients (70%) who had positivestaining in the sample of 40 patients with serous cyst-adenomas. Thirty-four (85%) and 78 (98%) had positive

staining in 40 borderline and 80 malignant seroustumor samples, respectively, but strong staining (3+)was present in 50% (n = 40) of the malignant versus 7%in the borderline group (Fig. 2). Their intensity of stain-ing conformed to the progress of serous ovarian tumor(P = 0.00) (see Table 1).

At the same ,time the finding shows positive corre-lation between GLUT1 and p63 expression (P = 0.00)(see Table 2).

Expression of GLUT1 and p63 according toclinicopathological characteristics of ourpatients with borderline and malignantserous ovarian tumor

Clinicopathological characteristics of our patient popu-lation are shown in Table 3, GLUT1 expression levels

a b

c d

Figure 1 Photomicrographs showing the differences inpositive staining for glucose transporter protein 1(GLUT1) in serous ovarian tumor. (a) Normal ovariantissues (negative) (GLUT1, original magnification×100). (b) Ovarian cystadenomas (weakly positive)(GLUT1, ×100). (c) Borderline serous ovarian cystad-enomas (moderately positive) (GLUT1, ×100). (d)Serous ovarian cystadenocarcinomas (strongly posi-tive) (GLUT1, ×250).

Table 1 GLUT1 and p63 expression differences in serous ovarian tumor group

n GLUT1 p63– 1+ 2+ 3+ P – 1+ 2+ 3+ P

Normal ovarian 40 40 0 0 40 0 0 0Serous ovarian tumor

Benign 40 13 27 (66%) 0 0 0.00*0.00**

12 24 (60%) 4 0 0.00*0.00**Borderline 40 0 18 22 0 6 18 13 3 (7%)

Malignant 80 0 0 15 65 (81%) 2 4 34 40 (50%)

*χ2-Test. **Spearman’s rank correlation coefficient. GLUT1, glucose transporter protein 1.

a b

c d

Figure 2 Photomicrographs showing the differences inpositive staining for p63 in serous ovarian tumor. (a)Normal ovarian tissues (negative) (p63, original mag-nification ×100). (b) Ovarian cystadenomas (weaklypositive) (p63, ×250). (c) Borderline serous ovarian cyst-adenomas (moderately positive) (p63, ×250). (d) Serousovarian cystadenocarcinomas (strongly positive)(p63, ×400).

Ovarian tumor

© 2014 The Authors 1927Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology

show statistical association with patient age, intraperi-toneal implants, ascites, lymph node status and Inter-national Federation of Gynecology and Obstetrics stage(FIGO) of malignant serous ovarian tumor. That is,there is statistical significance between GLUT1 expres-sion and malignant degree of tumor (P = 0.000),patients’ age (P = 0.037), FIGO stage (P = 0.000), intra-peritoneal implants (P = 0.002), ascites (P = 0.039) andlymph node status (P = 0.009); while only FIGO stage(P = 0.003), intraperitoneal implants (P = 0.030) andlymph node status (P = 0.003) have a statistical signifi-cance with the expression of p63. Patient age (P = 0.210)and ascites (P = 0.491) have no statistical associationwith the expression of p63 levels.

Discussion

The experiments show that the staining of the GLUT1transporter was completely negative in 40 normalovarian tissues. In 40 serous cystadenomas, weaklypositive expression of GLUT1 (1+) was present in 66%(n = 27), however, GLUT1 expression in 40 borderlineserous cystadenomas and 80 serous cystadenocarcino-mas was 100% positive, but it is clear that strong stain-ing (3+) was present in 81% (n = 65) of the malignanciesversus 0% in the borderline group. That is, there is aloss of GLUT1 expression in the normal ovarian seroustissues, but in the benign, borderline and malignanttumors the expression of GLUT1 is 66%, 100% and100%, respectively. The results are consistent with thereported paper. With the accelerated growth or malig-nant transformation of tumor cells, glucose transportand uptake are increased, and GLUT1 expressed in thetumor cells provides a reliable indicator for the identi-fication of borderline and malignant tumor. This studyalso showed that the expression of GLUT1 was posi-tively correlated with the clinical staging, intraperito-neal implants, ascites and lymph node metastasis,which is similar with Kalir et al.1 The relation of thedevelopment of ovarian serous cystadenocarcinoma

and the expression of GLUT1 will also be valuable forthe prognosis of patients with tumor.

This study accords with some results such as an asso-ciation between GLUT1 expression and neoplastic pro-gression in the natural history of endometrial andcolon carcinoma, esophageal squamous cell carci-noma;4 and conforms to the recent studies that haveshown that high GLUT1 expression is correlated with agreater frequency of lymph node metastases and a sig-nificant increase in mortality in patients with coloncarcinoma.4 Perhaps it is because GLUT1 is the mostimportant carrier of mammalian glucose transportacross the cell membrane into the cell, and is closelyrelated to the normal physiological process of theprotein membrane metabolism. It is not only involvedin the physiological glucose transport across the cellmembrane, but is also abnormally present in manypathological conditions. GLUT1 is higher in the redblood cells, brain capillary endothelium (BBB), cellmembrane perineurium and squamous epithelial basalcells, while in the other normal tissues it is undetect-able by immunohistochemical method. With the accel-erated cell growth or malignant cell transformation,glucose uptake and transport is increased, whichinduces abnormal expression of GLUT1, as has beenshown in a report regarding the expression of GLUT1in esophageal cancer, colorectal cancer and renal cellcarcinoma.2,13

This study shows that the p63 expression in thebenign, borderline and malignant ovarian seroustumor were 70%, 85% and 98%, respectively, and thedifference was statistically significant (P < 0.01). At thesame time, along with the increase of the clinicalstages, intraperitoneal implants and lymph nodemetastasis in ovarian serous carcinoma, the positiveexpression rate of p63 was higher, and the differencehad statistical significance, and was positively corre-lated (P < 0.05). However, patient age (P = 0.210) andascites (P = 0.491) are not in statistical association withthe expression of p63 levels. Through the abovedescription, it is evident that p63 is a marker for thedetection of the development and malignant progres-sion of ovarian serous tumor. The rate of p63 proteinexpression in ovarian serous cystadenocarcinomareached 98%, the lack of p63 in normal ovarian tissues,suggesting that p63 is a cancer gene candidate ratherthan tumor suppressor gene. This result and the DiComo et al.12 report shows that p63 is mainly expressedin basal cell and squamous cell carcinoma and trans-formed cells, but not in adenocarcinoma (e.g. prostatecancer, breast cancer, bladder cancer).14 The discovery

Table 2 Correlation between GLUT1 and p63 expression

n p63 rs

200 – 1+ 2+ 3+ P

GLUT1 – 53 45 8 0 0 0.819 (0.00)1+ 45 9 28 5 32+ 37 4 8 23 23+ 65 2 2 23 38

GLUT1, glucose transporter protein 1.

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1928 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology

that the lack of p63 accelerated cell apoptosis is consis-tent with Keyes et al.15 Similar results have been shownby Yao et al.16 p63 regulates a wide range of target genesthat are important for embryological development andepithelial integrity. Mutations of the p63 gene causeepidermal abnormalities characterized by ectodermaldysplasia. Recent reports have indicated that p63 playsan important role in tumorigenesis as well. However,the relative importance of p63 in ovarian serous tumordevelopment and tumorigenesis remains mostlyunclear and awaits further investigation.

The experimental results show that the positiveexpression of GLUT1 and p63 correlate with the occur-rence and development of tumor, and that the signifi-cant increase in expression may contribute to thecarcinogenicity. In conclusion, the experimental resultsshowed that GLUT1 and the p63 gene play a major rolein the occurrence and development, malignant trans-formation, invasion and metastasis of ovarian seroustumor, and may be used as markers of ovarian seroustumor malignant transformation, and also provide atheoretical basis for the diagnosis, prevention andtreatment of ovarian serous tumor. However, the latterwill need further study.

Acknowledgment

Professor Li Ying in the Department of PathologyBeijing TongRen Hospital, Capital Medical University,Beijing, China, provided technical support.

Disclosure

The authors have no conflict of interest to declare.

References

1. Kalir T, Wang BY, Goldfischer M et al. Immunohistochemicalstaining of GLUT1 in benign, borderline, and malignantovarian epithelia. Cancer 2002; 94: 1078–1082.

2. Wood IS, Trayhurn P. Glucose transporters (GLUT andSGLT): Expanded families of sugar transport proteins. Br JNutr 2003; 89: 3–9.

3. Zhdanov AV, Dmitriev RI, Papkovsky DB. Bafilomycin A1activates HIF-dependent signalling in human colon cancercells via mitochondrial uncoupling. Biosci Rep 2012; 32: 587–595.

4. He L, Li X, Luo HS et al. Possible mechanism for the regula-tion of glucose on proliferation, inhibition and apoptosis ofcolon cancer cells induced by sodium butyrate. World JGastroenterol 2007; 13: 4015–4018.

Table 3 Expression of GLUT1 and p63 according to clinicopathological charac-teristics of our patient with borderline and malignant serous ovarian tumor

Characteristics n GLUT1 p631+ 2+ 3+ P* – 1+ 2+ 3+ P*

Serous ovarian tumorBorderline 40 18 22 0 0.000 6 18 13 3 0.000Malignant 80 0 15 65 2 4 34 40

Age, years<35 19 6 11 2 0.037 2 4 13 0.21035–55 75 10 20 45 4 16 24 31>55 26 2 6 18 2 2 10 12

FIGO stageI 8 0 4 4 0.000 0 2 6 0 0.003II 13 0 6 7 0 0 10 3III 51 0 5 46 2 2 18 29IV 8 0 0 8 0 0 0 8

Intraperitoneal implantsNo 47 16 16 15 0.002 4 12 25 6 0.030Yes 73 2 21 50 4 10 22 37

AscitesNo 26 10 5 11 0.039 4 8 7 7 0.491Yes 64 6 26 32 4 10 28 22Cell+ 30 2 6 22 0 4 12 14

Lymph node statusNo 93 18 34 41 0.009 8 22 41 22 0.003Yes 27 0 3 24 0 0 6 21

*χ2-Test. FIGO, International Federation of Gynecology and Obstetrics; GLUT1, glucosetransporter protein 1.

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5. Hunnissen E, Boers E, Heideman DA et al. Correlation ofimmunohistochemical staining p63 and TTF-1 with EGFRand K-ras mutational spectrum and diagnostic reproducibil-ity in non small cell lung carcinoma. Virchows Arch 2012; 461:629–638.

6. Kaelin WG Jr. The p53 gene family. Oncogene 1999; 18: 7701–7705.

7. Brinck U, Ruschenburg I, Di Como CJ et al. Comparativestudy of p63 and p53 expression in tissue microarrays ofmalignant melanomas. Int J Mol Med 2002; 10: 707–711.

8. Aiad HA, Abd El-Halim Kandil M, Abd El-Wahed MM et al.Diagnostic role of p63 immunostaining in fine needle aspira-tion cytology of different breast lesions. Acta Cytol 2011; 55:149–157.

9. Cantuaria G, Fagotti A, Ferrandina G et al. GLUT-1 expressionin ovarian carcinoma: Association with survival and responseto chemotherapy. Cancer 2001; 92: 1144–1150.

10. Cai Y, Shao SL, Wang QH et al. Expression and significance ofGLUT-1 and DNA-PKcs in serous ovarian tumors. Ai Zheng2007; 26: 1188–1193.

11. Cantuaria G, Magalhaes A, Penalver M. Expression ofGLUT-1 glucose transporter in borderline and malignantepithelial tumors of the ovary. Gynecol Oncol 2000; 79: 33–37.

12. Di Como CJ, Urist MJ, Babayan I et al. p63 expression profilesin human normal and tumor tissues. Clin Cancer Res 2002; 8:494–501.

13. Kalir T, Rahaman J, Hagopian G et al. Immunohistochemicaldetection of glucose transporter GLUT1 in benign and malig-nant fallopian tube epithelia, with comparison to ovarian car-cinomas. Arch Pathol Lab Med 2005; 129: 651–654.

14. Stepan A, Margaritescu C, Simionescu C et al. E-cadherin andp63 immunoexpression in dysplastic lesions and urothelialcarcinomas of the bladder. Rom J Morphol Embryol 2009; 50:461–465.

15. Keyes WM, Wu Y, Vogel H et al. p63 deficiency activates aprogram of cellular senescence and leads to acceleratedaging. Genes Dev 2005; 19: 1986–1999.

16. Yao JY, Chen JK. Roles of p63 in epidermal development andtumorigenesis. Biomed J 2012; 35: 457–463.

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1930 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology