Comparative Study On The Expression Of Pim-3Related Molecules Between Gastric Cardia Adenocarcinoma And Distal Gastric Carcinoma

Objective：Gastric cancer continues to be a significant health issue, it issecond only to lung cancer as a leading cause of cancer deaths worldwide.During the past decades, the incidence of gastric cancer has been decreasedwith the change of the lifestyles, but the subsites of gastric carcinomaunderwent significant changes.The incidence of adenocarcinoma at antrum ordistal stomach has decreased, whereas that at esophagogastric junction （EGJ:the proximal stomach and distal esophagus） has increased in most developedcountries. Similar changes in the incidence of cardia and distal gastricadenocarcinoma in southern and central rural areas in Hebei province of Chinawere also found, but reasons were still unclear.Tumorigenesis and progression of gastric carcinoma are multistageprocesses accompanied with changes in gene expression. The pim family is agroup of oncogenes which encode the serine/threonine protein kinase. It iscomposed of pim-1, pim-2and pim-3.The serine/threonine kinase Pim isknown to promote cell cycle progression and to inhibit apoptosis leading totumorigenesis. They are extensively distributed in different tissues and organs,and play an important role in the growth, development of the body and celldifferentiation. Up to now, the crystal structure of the pim-3protein has notyet been obtained, but several independent groups have reported the crystalstructure of pim-1and pim-2in the presence or absence of theirinhibitors.Pim-3protein shows a high degree of identity with humanpim-1（57.1%） and pim-2（44.0%） at the amino acid level. Pim-3,a newmember of the family, can regulate the cell cycle, apoptosis andcarcinongenesis by phosphorylating specific substrates. Meanwhile, theupregulation of the pim-3gene can lead to the occurrence of the malignant tumors.Similar to that of other members of the pim kinase family, Pim kinasepromote cell cycle progression and tumorigenesis by down-regulating p27^kip1expression at both transcriptional and posttranslational levels. It is identifiedthat the CDK inhibitor p27^kip1as a novel substrate of pim kinases. P27^kip1isknown to be a key molecule that modulates cell cycle progression at the G1-Stranstition. Low p27^kip1expression has consistently been observed in manyforms of cancer in humans and correlates with tumor progression and possiblywith reduced survival in patients with cancer. Up to5%express p27, mainlybecause of accelerated proteolysis. It was also demonstrated that the stabilityof p27are regulated through direct phosphotylation.Ser10is the majorphosphotylation site of p27（phosphated Ser10P27^kip1）,accounting for70%of the total phosphorylation of the protein, due to cytolasmic localization andprogression to S phase. The Ser10phosphorylated p27accumulates in the cellcytoplasm during tumor development process.Expression profile analysis has demonstrated that pim-1contributes tothe regulation of approximately20%of Myc-regulated gene s. Moreover,Pim-1and Pim-2phosphorylate c-myc protein at its serine and threonineresidues. The results in stabilization and subsequent enhancement of thetranscription activities of c-myc protein.Furthermore, Pim-3can enhancec-myc mRNA expression via activation of PGC-1a. Thus, Pim kinases canpromote tumorigenesis by modulating the activities of c-Myc.Proliferation depends on the ability of the cell to successfully passthrough the G₁, S, G₂, and M phases of the cell cycle. A checkpoint at theG2/M boundary is known to play a role in cell cycle progression. The mostimportant protein regulating G₂/M transition is cyclin-dependent kinase1（cdc2）.It forms a complex with cdc25c, and enables cells to enter into the Mphase through the G₂/M boundary. Previously reported results suggest thatpim-1plays a role at the G2/M transition phase of the cell cycle by altering theactivity of cdc25c in two ways.First, pim-1activates the cdc25c phosphatasedirectly through phosphorylation, very probably at the N-terminal part of the protein.Second, pim-1blocks the C-TAk10mediated phosphorylation ofcdc25c on serine216that is inhibitory for cdc25c as described before. HumanPim-3shows a high sequence similarity with human Pim-1（57.1%）.It istempting to speculate that pim-3may also interact with cdc25c and cdc2.Therefore, we chose to further investigate pim-3, p-p27, c-myc, cdc2andcdc25c, five components of these signaling pathways in this study. To date,however, the expressions of pim-3, c-myc, p-p27, cdc2and cdc25c at proteinlevel and their potential biological roles in the tumorigenesis between gastriccardia and distal gastric adenocarcinoma of Chinese patients have not beenelucidated.Recent studies have suggested cardiac adenocarcinoma （GCA） tends tohave a greater tendency towards deeper wall penetration, more lymph nodemetastasis, and poorer prognosis than gastric cancer at other sites. It isreported that gastric cancers might be a special subtype or an independententity of gastric carcinoma. Thus, early diagnosis and early treatment areparticularly important for improving the survival rate. Up to now, few studiesabout the incidence and putative differences between adenocarcinomas ofdifferent sites of gastrc cancer were conducted in domestic and overseas, andthe molecular genetics changes between them are still quite few. Thus, it isvery necessary to find the differences in the expression of oncogene and tumorsuppressor gene between gastric cardia adenocarcinoma and distal gastriccarcinoma，and help to explore the possible carcinogenesis of gastric cardiaadenocarcinoma.In this study, immunohistochemical assays were used to determine theexpression rates of both GCA and DGA, pim-3, p-p27, c-myc, cdc2andcdc25c in140gastric cancer samples and their adjacent normal tissuespecimens by immunohistochemical. Additionally, the potential associationsbetween the expression patterns of the five markers and their associations withthe clinico-patholigical features of gastric cancers patients were evaluated.Methods:20cases of normal gastric mucosa（NGM）,78cases of gastriccardia adenocarcinoma（GCA） and62cases of distal gastric carcinoma（DGA） were studied the expression of pim-3, p-p27, c-myc, cdc2and cdc25c withimmunohistochemical method respectively. We also compared the relationshipbetween their expression with the clinical pathological features of GCA andDGA. The experimental datas were anlyzed with Chi-square test, Fisher’sexact test and correlation with stastics software of SPSS16.0edition.Results:1Immunohistochemical staining results of pim-31.1Expressions of pim-3in GCA and DGAExpressions of pim-3positive immunoreaction were located in cytoplasm asbrown granule. The positive expression rate of pim-3in GCA and DGA was64.1%and80.6%respectively. The incidence of pim-3positive expression inGCA was significantly lower than that in DGA（64.1%vs80.6%，P<0.05）.Among the20cases of normal gastric mucosa,4cases showed positive pim-3expression （20.0%）,which were all significantly lower than that in GCA andDGA （P<0.05）.1.2The clinical pathological significance of pim-3expression in GCA andDGAThe positive expression rate of pim-3in GCA was reltated with tumordifferentiation and tumor stage. The positive expression rate of pim-3inwell/moderate-differentiated group （38.5%） was significantly lower than thatin low-differentiated group （76.9%, P<0.05）.It was higher in cases with stageIII+IV than those with stageI+II （75.6%vs48.5%, p<0.05）. No relationshipbetween the positive expression of pim-3in GCA and the age and sex of thepatients, tumor size and lymph node metastasis was found （P>0.05）.In DGA, pim-3immunoreaction was observed more frequently in caseswith poor differentiation than that in well/moderate differentiation cases（89.1%vs56.3%, p<0.05）, in cases of advanced tumor stage（T3/T4） thanthose of early tumor stage （T1/T2）（94.0%vs65.5%, p<0.05）,and in cases withlymph node metastasis than those without lymph node metastasis（94.6%vs60.0%, p<0.05）,and there was no relationship between the expression of pim-3and the age and sex and the tumor size of the patients（P>0.05）. Thus, the results suggested that the significance of pim-3expression wasdifferent in GCA and DGA. The positive expression of pim-3in DGA wassignificantly related with lymph node metastasis, but no such relation wasseen in GCA.The incidence of pim-3positive expression in both DGA andGCA was significantly related with differatiation and tuomor stage.2Immunohistochemical staining results of p-p272.1Expressions of p-p27in GCA and DGAPositive immunohistochemical staining products were located in nucleus.The positive expression of p-p27in DGA was significantly higher than that inGCA （61.3%vs42.3%, P<0.05）,1case among the20cases of normal gastricmucosa showed positive pim-3expression（20.0%）,which are all significantlylower than that in GCA and DGA （P<0.05）.2.2The clinical pathological significance of p-p27expression in GCA andDGANo statistically significant relationship was found between p-p27expression and the age and sex of the patients, differatiation, size, clinicalstage and lymph node metastasis in GCA （P>0.05）.The positive expression of p-p27was significantly related withdifferentiation, clinical stage and lymph node metastasis in DGA. The positiveexpression in cases with well/moderated differentiation was significantlylower than that with poor differentiation group （37.5%vs69.6%, P<0.05）. Thepositive expression of p-p27was significantly higher in cases with stageIII+IV than that in cases with with stageI+II （78.8%vs41.4%, P<0.05）. Theincidence of p-p27positive expression were significantly higher in DGA withlymph node metastasis than those without lymph node metastasis （73.0%vs44.0%, p<0.05）.The expression of p-p27in DGA was not associated with ageand sex of the patients and size of tumor （P>0.05）.The positive expression of p-p27in DGA was closely related withdifferentiation, tumor stage and lymph node metastasis. However, norelationship was observed between p-p27expression and differentiation andtumor stage in GCA. 3Immunohistochemical staining results of c-myc3.1Expressions of c-myc in GCA and DGAPositive immunohistochemical staining products were located innucleus.The positive expression rate of c-myc in GCA and DGA was42.3%and66.1%respectively, which were all significantly higher than that inNGM （15.0%, P<0.05）.3.2The clinical pathological significance of c-myc expression in GCA andDGANo statistically significant relationship was found between c-mycexpression and the age and sex of the patients, differatiation, size, clinicalstage and lymph node metastasis in GCA （P>0.05）.The incidence of c-myc positive expression were significantly higher inDGA with lymph node metastasis than those without lymph node metastasis（78.4%vs48.0%, p<0.05）, as well as the higher expression in cases with stageIII+IV than those with stageI+II （84.8%vs44.8%, p<0.05）, while nostatistically significant relationship was found between c-myc expression andthe age and sex of the patients, defferentiation and size of tumor （P>0.05）.The positive expression of c-myc in DGA was closely related with tumorstage and lymph node metastasis, while no such relation was seen in GCA.4Immunohistochemical staining results of cdc24.1Expressions of cdc2in GCA and DGAPositive immunohistochemical staining products were located in nucleusas brown granules. The positive expressions of cdc2was detected in58（74.4%）of GCA and45（72.6%） of DGA, which were all significantly higher than thatin NGM （25.0%, P<0.05）.4.2The clinical pathological significance of cdc2expression in GCA andDGANo statistically significant relationship was found between cdc2expression and the age and sex of the patients, differatiation, size,clinical stageand lymph node metastasis in GCA and DGA （P>0.05）.5Immunohistochemical staining results of cdc25c 5.1Expressions of cdc25c in GCA and DGAAmong140cases with cardiac cancers, the positive expressions of cdc25cwas detected in67（85.9%） of GCA and46（74.2%） of DGA. However, thestaining data showed that protein expression is weakly detect in the normalgastric mucosa epithelium （30.0%）.5.2The clinical pathological significance of cdc25c expression in GCA andDGANo statistically significant relationship was found between cdc25cexpression and the age and sex of the patients, differatiation, size, tumor stageand lymph node metastasis in GCA and DGA （P>0.05）.6Corelation among pim-3, p-p27, c-my, cdc2and cdc25c expressions in GCAand DGAPositive correlation between expression of pim-3and p-p27was found inGCA and DGA（r=0.316, P=0.005; r=0.533, P=0.000）.There was positivecorrelation between expression of p-p27and c-myc in GCA and DGA（r=0.370, P=0.001; r=0.481, P=0.000）. Positive correlation could be foundbetween pim-3and c-myc in GCA（r=0.424, P=0.000） and DGA （r=0.598,P=0.000）.Conclusions：1The higher expression of pim-3, p-p27, c-myc, cdc2and cdc25c wasinvolved in GCA and DGA occurrence.2Significant difference was seen in the expression of pim-3, p-p27andc-myc between GCA and DGA. The expression of pim-3, p-p27and c-mycin GCA was lower than that in DGA. It is demonstrated that p-p27andc-myc regulated by pim-3play the different role both in GCA and DGA,but they effect more apparently in DGA.3No significant difference was seen in the expression of cdc2and cdc25cbetween GCA and DGA. It is suggested that both factors is not entirelydependent on the regulation of pim-3, p-p27and c-myc.4In DGA cases, the expression of pim-3, p-p27and c-myc was respectivelyrelated with differatiation, tumor stage and lymph node metastasis, While in GCA, only the expression of pim-3was related with differatiation andtumor stage.5The positive expression of pim-3in DGA was significantly related withlymph node metastasis, but no such relation was seen in GCA. The pim-3can be used as the risk of lymph node metastasis predictor in DGA.