The expression and meaning of Skp2，p27 proteins in Mucosa－associated lymphoid tissue lymphoma of ocular adnexal

Hong Lin，Li－Ting Hu，Gui－Qiu Zhao，Wen－Yi Yang，Jing－Jing Zhang

INTRODUCTION

Mucosa－associated lymphoid tissue(MALT)lymphoma，namely extranodal marginal zone B－cell lymphoma of MALT，is the most common type of ocular adnexal lymphoma and accounts for 10%－15% in the orbital solid tumors［1］.And the incidence has increased in recent years［2］. Most studies suggest that the majority of MALT lymphoma is lowgrade malignant B－cell lymphoma which rarely appears to be apt to metastasize and seriously deteriorate［3］.But at present，some foreign experts believe that extranodal lymphomas occurring in ocular adnexal is more aggressive［4］. Studies of the disease have asattracted many domestic and foreign scholars.The cell cycle inhibitory protein p27 may combine with cyclin directly to inhibit biological activity of cyclin－CDK complex，and high expression in G0phase to prevent cell shift from G1 to S phase，also inhibits cell cycle progress. Skp2 which is part of ubiquitin ligase complex can specifically recognize negative cell cycle regulatory factors，of which p27 is an important representative，can promote the progress of the cell cycle from G1 to S phase，and promotes cell proliferation.In recent years，the relationship between Skp2 and p27 increasingly attracts the attention of the researchers，but domestic research on both in the pathological changes of eye lymphoid tissue is not much.High expression of Skp2 and low expression of p27 may play a role in the occurrence and development of ocular lymphoma，and is associated with prognosis.This study detected the expression of Skp2 and p27 in MALT lymphoma by immunohistochemistry and combined with clinical data and pathological classification to discuss the effect of the two proteins on the prognosis of eye adnexal MALT lymphoma and explore their roles in the development of the diseases.

SUBJECTS AND METHODS

Subjects

Specimens collectionThe study included 20 specimens of ocular adnexal MALT lymphoma(which from 20 patients，8 from eyelid，10 from orbit and 2 from lacrimal gland)at the Pathological Lab of Ophthalmology Department，Affiliated Hospital of Medical College Qingdao University from 1995 to 2011.The ages of patients arranged from 52 to 88，with an average of 70.2±5.9.According to Ann Arbor clinical stage，the numbers of specimens from Ⅰto Ⅳphase were 16，2，1 and 1 respectively. 10 cases of reactive hyperplastic lymphadenopathy were used as positive control.For the use of these clinical materials for research purposes，the patient's consent and approval from the Institutional Research Ethics Committee were obtained.

HE and immumohistochemical stainingThe tissue from various stages was made to paraffin section and hematoxylineosin(HE)staining，and the tissue structure was observed under common microscope. 3－μm－thick paraffin sections were cut on a microtome(CM1900;Leica Microsystems，Deerfield，IL，USA)and then mounted onto glass slides with 100 g/L polylysine. Immunohistochemical staining was performed according to the manufacturer's instructions［SP kit(Biosynthesis Biotechnology)］.In brief，after deparaffinising in xylene and dehydrating in ethanol，the sections were immersed in a citrate buffer and heated in a microwave oven for 15min to retrieve antigens，then incubated with the mouse nonspecific immune serum(Liquid A)for 20min at room temperature. After that，the sections were incubated with either mouse anti－Skp2 polyclonal antibody(1:100)or mouse anti－p27 polyclonal antibody(1:40)(Biosynthesis Biotechnology)overnight at 4℃，and then incubated with horseradish peroxidase labeled goat anti－mouse polymer(Liquid B)for 30min at 37℃.After washing three times with PBS，diaminobenzidine was used as a chromogen，and haematoxylin was used as a counterstain. All specimens were sealed by neutral balsam for microscopy observation. As a negative control，the primary antibody was replaced with PBS.Specificity of primary antibody was validated by positive tests using reactive lymphoid hyperplasia. The positive expression is located in nucleus and sometimes in cytoplasm，which appears as diffuse brown yellow particles.

To avoid staining underestimation due to considerable regional variations，five representative fields were examined，and a total of 1000 cells(200 for each field)were counted under the microscope with a high power(×400)objective.Briefly，based on the scale of Xu et al［5］，the immunostaining grade was as follows:specimens in which ≤25%，＞25%－＜75%，≥75% of cells showed positive staining were defined as(－)，(+)，(++)respectively.

Statistical AnalysisStatistical analysis was performed by SPSS 17.0 software (SPSS，Chicago，IL，USA). The statistical differences of Skp2 and p27 expressions among the groups were determined by χ2tests，and among the stages were determined by Fisher's exact probabilities . The mutual relation between Skp2 and p27 was analyzed by Spearman correlation test. A P ＜0.05 was considered as statistically significant.

RESULTS

HE StainingThe samples of reactive lymphoid hyperplasia showed normal follicular structures with mature lymphocytes，whose patterns were various. It could be seen sporadic plasmocytes，histocytes and immunoblasts(Figure 1A). The samples of MALT lymphoma showed that high－differentiated and moderately－differentiated lymphoma was composed of diffuse，similar and small lymphocytes which appeared as circular nucleus，deep staining，little cytoplasm and extracellular matrix(Figure 1B).

Immumohistochemical Staining

Expression of Skp2in mucosa－associated lymphoid tissue lymphomaThere were no statistical differences in the expression rate of Skp2 between the groups with different Ann Arbor clinical stages，age，sex，occurrence site(P＞0.05，Table 1). The positive staining of Skp2 in MALT lymphoma and reactive lymphoid hyperplasia were mainly in nucleus，while there was no positive staining in the control groups.Moreover，the expression rate of Skp2 in MALT lymphoma(14/20，70.0%)(Figure 2A)was higher than that in reactive lymphoid hyperplasia (0/10)(Figure 2B)significantly(P＜0.05，Table 2).

Expression of p27in mucosa－associated lymphoid tissue lymphomaThere were no statistical differences in the expression rate of p27 between the groups with different Ann Arbor clinical stages，age，sex，occurrence site(P＞0.05，Table 1).The positive staining of p27 was mainly in nucleus.In reactive lymphoid hyperplasia，the positive staining of p27 was mainly located in small，mature and resting lymphocyte outside of germinal center(Figure 3A). The expression rate of p27 was low in tumor cells，while there were positive staining in normal small lymphocytes，mucosal epithelial cells and vascular endothelial cells in MALT lymphoma(Figure 3B).There was no positive staining in the control groups.The expression rate of p27 in MALT lymphoma (6/20，30.0%)was lower than that in reactive lymphoid hyperplasia(10/10，100%)significantly(P＜0.05，Table 2).

Table 1 Relationship between the expressions of Skp2/p27 and the clinical features of ocular adnexal MALT lymphoma n(%)

Table 2 The expression of Skp2 and p27 in MALT lymphoma and reactive lymphoid hyperplasia n(%)

Figure 1 HE Staining of reactive lymphoid hyperplasia and MALT lymphoma A:The samples of reactive lymphoid hyperplasia showed various cell patterns，sporadic plasmocytes，histocytes and immunoblasts. B:The samples of MALT lymphoma showed that high－differentiated and moderately－differentiated lymphoma was composed of diffuse，similar and small lymphocytes which appeared as circular nucleus，deep staining，little cytoplasm and extracellular matrix(HE×400).

Figure 2 Immumohistochemical Staining of Skp2 in MALT lymphoma and reactive lymphoid hyperplasia A:The expression rate of Skp2 in MALT lymphoma was high. The positive staining of Skp2 was mainly in nucleus. B:The expression rate of Skp2 in reactive lymphoid hyperplasia was low.The positive staining of Skp2 was mainly in nucleus(IH×400).

Figure 3 Immumohistochemical Staining of p27 in reactive lymphoid hyperplasia and MALT lymphoma A:The expression rate of p27 in reactive lymphoid hyperplasia was high. The positive staining of p27 was mainly in nucleus.B:The expression rate of p27 in MALT lymphoma was low. The positive staining of p27 was mainly in nucleus(IH×400).

Mutual relation between Skp2 and p27 in mucosa－associated lymphoid tissue lymphoma The results showed a negative relation between the expression of Skp2 and that of p27 in MALT lymphoma(r=－0.129，χ2=15.39，P＜0.05;Table 3).

DISCUSSION

Cell cycle regulation imbalance is considered to be a common feature of cell tumor. Cell cycle is regulated by multiple factors，of which the G1/S and G2/M are the two major turning points and respectively control cells into S and M phase. G1/S limit points are the key and rate－limiting stage to control the growth of the cells and determine their fate. Inrecent years，with the development of molecular biology，much attention is attracted to the researches on cell cycle regulatory protein. While the cell cycle regulatory protein is combined with the detection of the molecular markers of Skp2 andp27，it can provide important index and basis for the clinic and scientifically predict the patient's disease type and offer help to select the appropriate clinical treatment plan［6］.Skp2，found in recent years，is a kind of F－box protein. It functions as a recognition subunit to the substrates of SCF complex. It can specifically recognize 187th threonine phosphorylated p27 and mediate its poly－ubiquitination and degradation.Skp2 must compound with other subunits，such as Cull，Rbxl，Skpl to form SCF－Skp2 compounds to play a role，mediate poly－ubiquitination reaction. Abnormity of this process directly affects the absolute content of intracellular p27 protein，promotes the progress of the cell cycle from G1 to S phase，and promotes cell proliferation［7，8］.Skp2 appears in G0 advanced stage and acts on S phase. If Skp2 expression increased，p27 expression would reduce，these would allow the tumor cells to constantly pass through the Go/S regulation points to enter the cell cycle and lead to overgrowth and malignant proliferation of the cells and tumor formation［9］.Previous studies had shown that Skp2 overexpresses in many human cancer tissues and high expressions are often associated with the occurrence of tumor［10－12］.In this study，we detected that the positive rate of Skp2 in MALT lymphoma was 70.0%(14/20)，which is similar to the result of Shigemasa et al［13］. But there was no expression of Skp2 in reactive lymphoid hyperplasia，which suggests that high expression of Skp2 is associated with the occurrence of MALT lymphoma.Skp2 protein was highly expressed in ocular adnexal MALT lymphoma，suggesting that such indicator has obvious heterogeneity and capable to be used as important basis of diagnosis and differential diagnosis for ocular adnexal MALT lymphoma.Meanwhile，the study showed that the expression of Skp2 protein was not related with patients'Ann Arbor staging，which was different from the point of White et al［14］.And there was not obvious correlation with age，gender and occurrence site.

p27 n －Skp2+ ++14 1 5 8+ 5 3 2 0++ 1 1 0 0

p27 is a member of the family of cKip/ciP，which is the first to inhibit cyclin E/CDK 2 and cyclin A/CDK 2 complex activity. Cell cycle protein E－CDK2 is the key to the cells to through G1/S limit point，P27 stagnates cells in G1 phase by inhibiting cell cycle protein E－CDK2. As Gl period level checkpoint related gene，p27，in the normal cell cycle，expresses as a curve，and in G0 and G1 phase to achieve highest，once the cells come into the cell cycle，lower levels of p27 may maintain the stability of the phase of the cell in G0.Therefore deficiency of p27 protein will inevitably lead to cell cycle process accelerated，have close relationship with the occurrence of tumor development［15，16］.

The expression of p27 in MALT lymphoma was analyzed. It showed that the average expression level of p27 protein in lymphoid tumor cells was significantly lower than that in reactive lymphoid hyperplasia(outside of germinal centers).Compared with reactive lymphoid tissue hyperplasia，the positive rate of p27 protein declined，which suggested that low expression of p27 might be associated with the occurrence of MALT lymphoma and it was in agreement with the study of M Kiviniemi［17］.P27 protein of Ocular adnexal MALT lymphoma was in low expression. Meanwhile，the study showed that the expression of p27 protein was not significantly related with patients'Ann Arbor staging，and not with age，gender and occurrence site.

It is generally believed that Skp2 expression is negatively correlated with p27［18］.F－box protein Skp2 is the recognition subunits of SCFSkp2 composite substrate［19］. It can play a role to mediate ubiquitination reaction only if Skp2 and other subunits combine to form complexes. Thrl87 phosphorylation is the necessary condition for Skp2 to identify the ubiquitin degradation of p27 and it can directly weaken the degradation pathway mediated by Skp2 and lead to cell cycle arrest if the phosphorylation of the site is disturbed［20］. This study found that in MALT the expression of Skp2 and p27 was negatively correlated，which supports the point of Lahav－Baratz et al［21］.

The pathogenic mechanism of ocular adnexal MALT lymphoma is the joint action of multiple factors.This experiment suggests that decrease in p27 expression and increase in Skp2 expression may play a certain role in the development of MALT lymphoma.It was reported that high expression of Skp2 and lower levels of p27 give us a selection for drug therapeutic development by targeting these molecules. Continued investigations into the molecular mechanisms of MALT lymphoma may provide to establish novel biomarkers for this disease and generate therapeutic targets for improved treatment［22］.Comprehensive analysis of the expression of p27 and Skp2 protein may have certain guiding significance on the prognosis evaluation of the disease. The exact mechanism of how the expression of Skp2 and pP27 protein affects the prognosis is still required to be studied further，while the indepth discussion on the action mechanism of both will also provide a new idea for the treatment of ocular adnexal MALT lymphoma.

1 Harada K，Murakami N，Kitaguchi M，Sekii S，Takahashi K，Yoshio K，Inaba K，Morota M，Ito Y，Sumi M，Suzuki S，Tobinai K，Uno T，Itami J.Localized ocular adnexal mucosa－associated lymphoid tissue lymphoma treated with radiation therapy:a long－term outcome in 86

patients with 104 treated eyes. Int J Radiat Oncol Biol Phys 2014;88(3):650－654

2 Smiljanic M，Milosevic R，Antic D，Andjelic B，Djurasinovic V，Todorovic M，Bila J，Bogdanovic A，Mihaljevic B.Orbital and ocular adnexal Mucosa－Associated Lymphoid Tissue(MALT)lymphomas:a single－center 10－year experience.Med Oncol 2013;30(4):722

3 Adrish M，Venkatram S，Niazi M，Diaz－Fuentes G.Concurrent lung squamous cell carcinoma and extranodal marginal zone B－cell lymphoma of mucosa－associated lymphoid tissue type. J Bronchology Interv Pulmonol 2014;21(1):96－99

4 Lim SH，Kang M，Son J.Extranodal marginal zone B cell lymphoma of mucosa－associated lymphoid tissue type of the ocular adnexa:retrospective single institution review of 95 patients.Indian J Ophthalmol 2011;59(4):273－277

5 Xu HT，Li QC，Zhang YX，Zhao Y，Liu Y，Yang ZQ，Wang EH.Connexin 43 recruits E－cadherin expression and inhibits the malignant behaviour of lung cancer cells. Folia Histochem Cytobiol 2008;46(3):315－321

6 Kullmann MK，Grubbauer C，Goetsch K，J?kel H，Podmirseg SR，Trockenbacher A，Ploner C，Cato AC，Weiss C，Kofler R，Hengst L.The p27－Skp2 axis mediates glucocorticoid－induced cell cycle arrest in Tlymphoma cells.Cell Cycle 2013;12(16):2625－2635

7 Hatton BA，Ellison DW，Gajjar A，Kool M，F(xiàn)ero M，Olson JM.Prognostic value and functional consequences of cell cycle inhibitor p27Kip1 loss in medulloblastoma.Biomark Res 2013;1(1):14

8 Miyai K，Yamamoto S，Iwaya K，Asano T，Tamai S，Tsuda H，Matsubara O.Altered expression of p27(Kip1)－interacting cell－cycle regulators in the adult testicular germ cell tumors:potential role in tumor development and histological progression. APMIS 2012;120(11):890－900

9 Hsieh HY，Shieh JJ，Chen CJ，Pan MY，Yang SY，Lin SC，Chang JS，Lee AY，Chang CC.Prodigiosin down－regulates SKP2 to induce p27(KIP1)stabilization and antiproliferation in human lung adenocarcinoma cells.Br J Pharmacol 2012;166(7):2095－2108

10 Lv A，Li Z，Tian X，Guan X，Zhao M，Dong B，Hao C.SKP2 high expression，KIT exon 11 deletions，and gastrointestinal bleeding as predictors of poor prognosis in primary gastrointestinal stromal tumors.PLoS One 2013;8(5):e62951

11 Tosco P，La Terra Maggiore GM，F(xiàn)orni P，Berrone S，Chiusa L，Garzino－Demo P. Correlation between Skp2 expression and nodal metastasis in stage I and II oral squamous cell carcinomas. Oral Dis 2011;17(1):102－108

12 Hung WC，Tseng WL，Shiea J，Chang HC.Skp2 overexpression increases the expression of MMP－2 and MMP－9 and invasion of lung cancer cells.Cancer Lett 2010;288(2):156－161

13 Shigemasa K，Gu L，O'Brien TJ，Ohama K.Skp2 overexpression is a prognostic factor in patients with ovarian adenocarcinoma. Clin Cancer Res 2003;9(5):1756－1763

14 White WL，F(xiàn)erry JA，Harris NL，Grove AS Jr.Ocular adnexal lymphoma.A clinicopathologic study with identification of lymphomas of mucosa－associated lymphoid tissue type. Ophthalmology 1995;102(12):1994－2006

15 Miyamoto T，Horiuchi A，Kashima H，Suzuki A，Yamada T，Kurai M，Konishi I，Shiozawa T.Inverse correlation between Skp2 and p27(Kip1)in normal endometrium and endometrial carcinoma. Gynecol Endocrinol 2010;26(3):220－229

16 Huang KT，Pavlides SC，Lecanda J，Blank SV，Mittal KR，Gold LI.Estrogen and progesterone regulate p27kip1 levels via the ubiquitinproteasome system:pathogenic and therapeutic implications for endometrial cancer.PLoS One 2012;7(9):e46072

17 Kiviniemi M，Sauroja I，Rajam?ki A，Punnonen K，S?derstr?m KO，Salminen E.Cell cycle regulators p27 and pRb in lymphomas－correlation with histology and proliferative activity.Br J Cancer 2000;83(9):1161－1167

18 Yan L，Yun N，Xiu－Min D，Xu－Qi X. Oncogenic role of Skp2 and p27Kip1 in intraductal proliferative lesions of the breast.Chin Med Sci J 2012;27(3):161－166

19 Bornstein G，Ganoth D，Hershko A.Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F－box protein and substrate.Proc Natl Acad Sci U S A 2006;103(31):11515－11520 20 Guan X，Chen L，Wang J，Geng H，Chu X，Zhang Q，Du L，De W.Mutations of phosphorylation sites Ser10 and Thr187 of p27Kip1 abolish cytoplasmic redistribution but do not abrogate G0/1 phase arrest in the HepG2 cell line.Biochem Biophys Res Commun 2006;347(3):601－607 21 Lahav－Baratz S，Ben－Izhak O，Sabo E，Ben－Eliezer S，Lavie O，Ishai D，Ciechanover A，Dirnfeld M.Decreased level of the cell cycle regulator p27 and increased level of its ubiquitin ligase Skp2 in endometrial carcinoma but not in normal secretory or in hyperstimulated endometrium.Mol Hum Reprod 2004;10(8):567－572

22 Adams J，Elliott PJ. New agents in cancer clinical trials. Oncogene 2000;19(56):6687－6692