The Expression And Significance Of Pin1 And β-catenin In Squamous Cell Carcinoma And Adenocarcinoma Of Lung

IntroductionPhosphorylation of proteins on serine or threonine residues preceding pro-line ( Ser/Thr - Pro) is an important signaling mechanism controlling many cellular processes, such as cell cycle regulation, cell differentiation and proliferation. The conformation and function of phosphorylated Ser/Thr - Pro motifs are further regulated by the prolyl isomerase Pinl. β- catenin is a multifunctional protein that plays an important role in the transduction of Wnt signals and in the intercellular adhesion by linking the cytoplasmic domain of E - cadherin/ catenin complex. Interestingly, overexpression of Pinl can contribute to the aberrant expression of β - catenin in the nucleus, which enhances the transcription of a number of target genes,including the cyclinDl gene and Myc,which can lead to cell proliferation . Recent work indicates that Pinl is overexpressed in many human cancers and plays an important role in oncogenesis. Now there have been few reports about the expression of Pinl in lung cancer tissue and the relationship with β - catenin. So we investigated the expression of Pinl, β - catenin and the relationship of their expression in squamous cell carcinoma and adenocarci-noma of lung in order to evaluate their possible roles in the oncogenesis and progression of lung cancer.Materials and Methods1. Samples;69 lung cancer tissue samples (45 lung squamous carcinomas and 24 lung adenocarcinomas) . 49 lung cancer tissue samples among those were obtainedfrom patients who had surgery in the LiaoNing Province Tumor Hospital between 1980 and 2001. The other 20 fresh lung cancer tissue samples and 10 fresh non - cancerous tissues were obtained from the First Affiliated Hospital of China Medical University between March to July,2005.2. ReagentThe anti -human Pinl rabbit polyclonal antibody(SC - 15340) ,the goat -anti - mouse second antibody (ZB - 2305 ) , the goat - anti - rabbit second antibody (ZB -2301) , protein marker (P7708V)were bought from Santa Cruz company (USA ) . The anti - human (3 — catenin mouse monoclonal antibody ( ZM -0442 ) was provided by Zymed company ( USA) . The Ultro - sensitive S - P kit (SP -9000) and DAB agent kit(ZLI -9032) were bought from Beijing zhongs-han golden bridge biotechnology co. , LTD.3. Method3. 1 Immunohistochemistry: Detect the Pinl and [3 -catenin expression by immunohistochemistry S -P method. Reasult determination: CD Pinl;The normal staining was in the nucleus;the overexpressed staining was in the nucleus and/or cytoplasm. ( A ) The staining intensity was scorded as negative ( 0 ) , weak ( 1) , morderate ( 2 ) , and strong ( 3 );( B ) The number of cells stained was graded as follows: s￡25% (0) , 25% -50% (1) , 51% -75% (2) , =2 75% (3). AxB, Grades^3 showed its overexpression. (2)(3 - catenin: more than 70% cells showing membrane staining showed its normal expression. On the contrary, we called it membrane weak or negative staining . Both membrane weak or negative staining and more than 10% cells showing cytoplasmic and/or nucleal staining showed its aberrant expression.3. 2 Western Blot: Detect the expression of Pinl and ￡$ - catenin in lung cancer tissues and noncancerous tissues. Rapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer centrifuge the homogenate ( 10,000rpm, 4X.) for 60 minutes to pellet insoluble material. (l)Electrophoresis;(2)transfer proteins from gel to PVDF membrane;(3)blocking;(4)incubation with primary antibody;? enzyme conjugate incubation;?substrate incubation;?DAB staining.4. Statistical analysisAll the data were analyzed with SPSS for Windows 10.0 software. We usedX2test to analyze the correlation between clinicopathologic parameters of lung cancer and expressions of Pinl and p - catenin. We used the exact permutation test for the Spearman correlation coefficient to analyze the correlation between expression of Pinl and p - catenin. The results of Western Blot was analyzed by t test. A P value of less than 0.05 was accepted as statistically significant.Result1. Expression of Pinl in squamous cell carcimoma of lung, adenocarcinoma of lung and normal tissues.Pinl was overexpressed in lung cancer tissues and localized in nucleus and/or cytoplasm. Western Blot Method showed the level of Pinl in the tumor tissues is noticeably higher than that in the normal tissues(t = 15. 84, P = 0. 0000).2. Expression of p - catenin in squamous cell carcimoma of lung, adenocarcinoma of lung and normal tissues.P - catenin showed aberrant expression in lung cancer tissues, which was confined to the nucleus and/or cytoplasm, while the cell membrane showed weak or negative staining. Western Blot Method showed the level of fj — catenin in the tumor tissues is noticeably higher than that in the normal tissues (t = 13. 65 ,P = 0.0000).3. Clinical significance of Pinl and p - cateninOverexpression of Pinl and aberrant expression of p - catenin were found.in 54 (78. 3% ) and 44 (63. 8% ) of 69 lung cancer cases,respectively. Overexpression of Pinl and aberrant expression of p - catenin were found in 15 (75% ) and 16 (80% ) of 20 lung cancer cases, respectively, which were also analyzed by Western Blot. The expression of Pinl was not correlated to age ( P = 0. 0836) ,sex (P =0.5358) , histological classification (P =0.4557) ,differentiation (P =0. 6945 ) ,lymph node metasis (P = 0. 6645) and P - TNM stage (P = 0. 7783) . The expression of p - catenin was correlated to the differentiation ( P = 0.0223) ,lymph node metasis (P=0.0164) of lung cancer. Additionally, overexpression of Pinl and aberrant expression ofp - catenin showed a signifi-cantly positive correlation. (r = 0.447 , P = 0.0021)DiscussionPhosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr -Pro) is a major intracellular signaling mechanism. The phosphoryla-ted Ser/Thr - Pro motifs in a certain subset of phosphoproteins are isomerized specifically by thepeptidyl - prolyl cis - trans isomerase Pinl. This post - phosphorylation isomerization can lead to conformational changes in the substrate proteins and modulate their functions. Recent work indicates that Pinl is overex-pressedin many human cancers and plays an important role in oncogenesis.In the present study, we found that Pinl was overexpressed in lung cancer tissues, but that Pinl protein levels in lung cancer cells were not positively correlated with any commonly used clinicopathologic parameters, such as tumor stage and lymph node metastasis. Rescent work indicated that Pinl levels positively correlated with the tumor grade in invasive breast cancer and its levels were considerably higher in prostate cancers with poor survivable levels. These results demonstrate that Pinl may be only related to the oncogenesis not the progression and development of lung cancer. Since inhibition of Pinl induces apop-tosis , Pinl could represent a new anti - cancer target. Since E2F proteins bind the PIN1 promoterin vitro and in vivo, and increased Pinl levels in breast cancer cell lines correlate with an increase in the binding of E2F to the PIN1 promoter. Moreover, overexpression of E2F enhances PIN1 promoter activity and mR-NA levels in breast cancer cells. Therefore it is possible that deregulation of E2F plays a key role in the upregulationof Pinl in breast cancer. Since deregulation of the E2F/Rb pathway is also found in many other cancer types and contributes to the oncogenesis of a number of human cancers, deregulation of the E2F/Rb pathway may cause Pinl overexpression in lung cancer cells.P - catenin is a multifunctional protein that plays an important role in the transduction of Wnt signals and in the intercellular adhesion by linking the cyto-plasmic domain of E - cadherin/ catenin complex. Alterations of p - catenin expression have been documented in many malignancies and its levels were corre-lated with oncogenesis and progression of these cancers. We also found aberrant P — catenin expression in lung cancers, and the expression was related to the differentiation, lymph node metasis of lung cancer. Additionally, overexpression of Pinl can upregulate (3 - catenin in breast cancers, by inhibiting interaction between APC and p - catenin. Interestingly, we also found that p - catenin abnormally expressed in 40 of 44 (90. 9% ) cases with overexpressed Pinl, and statistical analysis revealed a significant correlation between Pinl overexpression and P — catenin expression , suggesting that Pinl overexpression is significantly correlated with p - catenin in lung cancer and further supports, that aberrant Pinl expression may be important to the upregulation of p - catenin expression. Our work indicates that Pinl is overexpressed in human lung cancers and aberrant Pinl expression may be important to the upregulation of p - catenin expression. Since Pinl regulates the expression of cyclin Dl by cooperating with Ras signaling and inhibiting the interaction of p - catenin with the tumor suppressor APC and also directly stabilizing cyclinDl protein, so Pinl may play an important role in oncogenesis. Given its role in cell growth control and oncogenesis , Pinl could represent a new anti - cancer target.Conclusion1. The overexpression of Pinl is associated with the oncogenesis of lung cancer.2. In lung cancer tissues, overexpression of Pinl and aberrant expression of P — catenin showed a significantly positive correlation.