Pin1 Overexpression In Squamous Cell Carcinoma And Adenocarcinoma Of Lung And Its Correlation With CyclinD1

Oncogenesis is a multistep and multifactorial process both at the genetic and epi - genetic levels, that results in uncontrolled cell proliferation, transformation, and cell death. One major regulatory mechanism in cell proliferation, transformation is phosphorylation of proteins on serine or threonine residues preceding proline ( pSer/Thr - Pro) by various prodirected protein kinases, such as cyclin - dependent kinases. The pSer/Thr - Pro motifs in proteins exist in two completely distinct cis and trans conformations, whose conversion is normally restrained by phophorylation, but catalyzed specifically by the essential prolyl isomerase Pin1. By isomerizing specific pSer/Thr - Pro bonds, Pinl has been shown to catalytically induce conformational changes in proteins after phosphorylation , thereby having profound affects on their catalytic activity, dephospho-rylation , protein - protein interactions, subcellular location. Thus, Pinl -dependent phosphorylation - specific prolyl isomerization is a critical new signaling mechanism.The level of Pinl in tumor tissues is noticeably higher than that in the normal tissues, and it keeps correlation to clinical stage, tumor stage. Now there have been little reports about the expression of Pinl in lung cancer tissue and the relationship with CyclinD1, so we investigated the expression of Pinl and Cy-clinDl in lung squamous carcinoma and adenocarcinomas cases with the neighboring noncancerous tissues, to analyze whether they were related to clinical pathological factors. Thus we can evaluate their role and significance in lungcancer generation and development.Materials and Methods1. Samples45 lung squamous carcinomas and 24 lung adenocarcinomas with neighboring non - cancerous tissue samples were obtained from patients who had surgery in the LiaoNing Province Tumor Hospital between 1980 and 2001 and the First Affiliated Hospital of China Medical University between September to November 2003, fixed in 4% paraformaldehyde, embedded in paraffin. 30 freshly tissue samples of them (10 lung squamous carcinomas N10 lung adenocarcinomas and 10 non -cancerous tissues) were prepared for Western Blot.The antibody Pinl polyclonal antibody was provided by Santa Cruz Company of America. The Ultro - sensitive S - P kit x DAB agent kit N the (3 - action antibody and the goat - and - rabbit second antibody were bought from ZhongS-han Biological Company of Beijing.3. Method3. 1. Immunohistochemistry;Detect the Pinl expression by immunohisto-chemistry S - P method. Result determination: (1) Pinl was a nucleus and ( or) cytoplasm staining protein. A: The staining intensity was scored as negative (0 v) , weak staining( V) , morderate or strong staining(3);B;The number of cell stained was graded as follows;0v, less than 25% of tumor cells showed immunore-activity;lv, 25% -49% of tumor cells showed immunoreactivity;2", 50% -74% of tumor cells showed immunoreactivity;3 v, over 75% of tumor cells showed immunoreactivity. A B, Grades over 3 were regarded overexpression. (2) CyclinDl was a nucleus and (or) cytoplasm staining protein. Less than 20% of tumor cells showed negative , and over 20% of tumor cells showed positive.3.2. Western Blot;Detect the expression of Pinl and CyclinDl in lung tissues and neighboring noncancerous tissues . Rapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer centrifuge the homogenate (10,000rpm,4T! ) for 60 minutes to pellet insoluble material . (l)Electrophoresis;(2)transfer from gel toPVDF membrane;(3) blocking;(4) incubation with primary antibody;(5) enzyme conjugate incubation;?substrate incubation.4. Statistical analysisAll the data were analyzed with PEMS 3. 1 medical statistics software, x ~ test was to compare the clinicopathological characteristics with the expression of Pinl and GyclinDl. The statiscal significance was defined as P<0.05.Result1. Expression of Pinl in lung squamous carcinomas %lung adenocarcinomas and normal tissuesPinl was weak or no expression in alveolar epithelial cells , and was strong expression in the nucleus and (or) cytoplasm of the lung squamous carcinomas N lung adenocarcinomas cells. Western Blot Method showed the level of Pinl in the tumor tissues is noticeably higher than that in the normal tissues.2. Expression of CyclinDl in lung squamous carcinomas .Jung adenocarcinomas and normal tissuesCyclinDl was strong expression in the nucleus and (or) cytoplasm of the lung squamous carcinomas xlung adenocarcinomas cells. Western Blot Method showed the level of Pinl in the tumor tissues is noticeably higher than that in the normal tissues.3. Clinical Significance of Pinl and CyclinDlThe expression of Pinl protein was not related to agexsexxhistological type% differentiation xlymph node metastasis and P -TNM stage. The expression of CyclinDl protein was related to differentiation ( P = 0. 0274) , but not related to ageNsexNhistological type ^ differentiation N lymph node metastasis and P - TNM stage. Pinl kept significant positive correlation to CyclinDl (p =0.0048).DiscussionPhosphorylation of proteins on certain serine/threonine residues preceding proline ( pSer/Ser - Pro) is a major mechanism in regulating cell proliferationand transformation. For example, signaling pathways triggered by the oncogenes Neu and Ras lead to the activation of various Pro - directed kinases, which e-ventually enhances transcription of the CyclinDl gene via multiple transcription factors, including E2F, c - jun/AP - 1, fj - catenin/TCF and NF - kB. Furthermore , CyclinDl stability is regulated by post - translation phosphorylation on the Thr286 - Pro. The conformation and function of phosphorylated Ser/Ser -Pro motifs are further regulated by the prolyl isomerase Pinl. Thus Pinl - dependent phosphorylation - specific prolyl isomerzation is an important new signaling mechanism.CyclinDl plays a pivoltal role in the development of cancer, its ablation suppresses the ability of Ha/Ras or c - Neu/HER2/EerB2 to induce cancer in mice. These results indicate that CyclinDl is an essential downstream target for tumorigenesis induced by oncogenic Neu or Ras, and that a major mechanism in these oncogenic processes is the phosphorylated Ser/Ser - Pro motifs. We have previously found Pinl overexpression in human cancer tissues, and its expression levels closely correlate with CyclinDl levels in cancer tissues. Importantly, upregulation of Pinl has been shown to elevate CyclinDl gene expression by activating c - jun/AP - 1 % p - catenin/TCF and NF - kB transcription factors. Pinl also directly bind to the phosphorylated Thr286 - Pro motif inhibiting its export into the cytoplasm. In addition the breast epithelial compartment in Pinl -/ - adult females failed to undergo the massive proliferation changes associated with pregnancy. Finally, Pinl transcription is enhanced by oncogenic Neu or Ras signling via E2F activation and it enhance the transformed phenotypes of Neu/Ras - transfected mammary epithelial cells. These results provide in vivo and ex vivo evidence for an essential role of Pinl in early events of tumorgenesis and strongly support Pinl as an attractive anticancer target.In the present study, Pinl and CylinDl are overexpression in lung squa-mous carcinomas and lung adenocarcinomas, and Pinl kept significant positive correlation to CyclinDl . our experiment confirm that Pinl is a critical factor in the Neu/Ras activated signal pathway by Cyclin Dl. So, Pinl is a essential factor in the development of cancer.Conclusion1. The level of Pinl in the tumor tissues is noticeably higher than that in the normal tissues. The level of CyclinDl in the tumor tissues is noticeably higher than that in the normal tissues.2. The expression of Pinl protein was not related to age % sex x histological type^differentiationxlymph node metastasis and P - TNM stage. The expression of CyclinDl protein was related to differentiation, but not related to agexsexxhis-tological type N differentiation N lymph node metastasis and P—TNM stage.3. Pinl kept significant positive correlation to CyclinDl.