Prevalent Overexpression of Prolyl Isomerase Pin1 in Human Cancers
Abstract
Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings that Pin1 expression is an excellent prognostic marker in prostate cancer and that inhibition of Pin1 can suppress transformed phenotypes and inhibit tumor cell growth, these findings may have important implications for the pathogenesis, diagnosis, and treatment of human cancers.
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 and transformation is phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) by various prodirected protein kinases, such as MAP kinases, cyclin-dependent kinases, JNK, and GSK3β.1–3 Interestingly, the pSer/Thr-Pro motifs in proteins exist in two completely distinct cis and trans conformations, whose conversion is normally restrained by phosphorylation, but catalyzed specifically by the essential prolyl isomerase Pin1.3–6 By isomerizing specific pSer/Thr-Pro bonds, Pin1 has been shown to catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on their catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover.4,5,7–18 Thus, phosphorylation-dependent prolyl isomerization is a critical postphosphorylation regulatory mechanism in phosphorylation signaling.3
Recently, it has been reported that Pin1 is overexpressed in human breast cancer cell lines and breast cancer tissues, and its expression closely correlates with the level of cyclin D1 in tumors.15 Furthermore, Pin1 positively regulates cyclin D1 function at the transcriptional level by activating β-catenin/TCF transcription factors and c-jun/AP-1 transcriptional factors, and also through posttranslational stabilization.15–17 Moreover, Pin1 is an E2F downstream target gene, whose expression is activated by various oncogenic proteins such as Neu and/or Ras.18 In addition, the transient conformational change of BclII because of association with peptidyl prolyl isomerase might contribute to apoptotic signaling,19,20 and the transcription factor nuclear factor-κB signaling is also regulated by Pin1.21 Overexpression of Pin1 not only confers transforming properties on normal mammary epithelial cells, but also enhances transformed phenotypes of Neu/Ras-transformed mammary epithelial cells.18 In contrast, inhibition of Pin1 suppresses the Neu- and Ras-induced transformed phenotypes or induces tumor cells into mitotic arrest and apoptosis.18 Consistent with a role of Pin1 in cell growth regulation, Pin1 knockout mice displayed a range of cell proliferative abnormalities, including decreased body size, testicular atrophy, retinal degeneration, and neurological abnormality.14 Moreover, in Pin1 adult female mice the breast epithelial compartment failed to undergo the massive proliferative changes associated with pregnancy,17 indicating that Pin1 is critical for cell proliferation in vivo. Finally, we have recently shown that Pin1 expression is positively correlated with clinical stage in human prostate cancer.22 Of 580 prostate cancer patients analyzed, patients with a higher level of Pin1 expression have a significantly higher probability of recurrence than their counterparts with low Pin1 expression after radical prostatectomy, even when patients with Gleason score 6 or 7 are analyzed separately. Together, these results strongly suggest that overexpression of Pin1 may promote tumor cell growth and contribute to the malignancy of cancer cells.
Despite the evidence that Pin1 protein is overexpressed in human breast cancers and its significant prognostic role in prostate cancer, there have been no previous reports that have addressed Pin1 protein levels in other human cancerous tissues. In the present study we conducted a quantitative investigation on 2041 human tumor samples and 609 normal tissue samples as well as 10 human transformed cell lines and three human normal cell lines to investigate the levels of Pin1 protein in various tumor tissues and compared that with corresponding normal tissues. Consideration of whether Pin1 protein is overexpressed in cancer versus normal tissues will provide valuable insights into the significance of Pin1 in oncogenesis and eventually this may be useful for development and application of more effective and potentially curative treatment strategies in which the inhibition of Pin1protein is an integral component.
WI 38, HUV-EC-C, and MCF10a are cell lines derived from human normal tissues. SW 620, PC-3, DU 145, RPMI 7951, T47D, MDA-MB-435, MCF7, T98G, DBTRG 05MG, and SW1271 are cell lines derived from human tumor tissues. Pin1 concentrations as determined from Pin1:actin ratios are averages from a minimum of three gels.
++, Moderate, +++, strong.
Normal(*) indicates that the normal tissues were obtained from cancer patients. Tumor lysates are identified by patient number. Both normal and cancer tissue were obtained from patients 874 and 910. Pin1 concentrations, as determined by immunoblotting analysis with the anti-Pin1 monoclonal antibody, were expressed as average Pin1:actin ratios from multiple Western blots.
Footnotes
Address reprint requests to Da-Gong Wang, Pintex Pharmaceuticals Inc., 313 Pleasant St., Watertown, MA 02472. .moc.mrahpxetnip@gnawgd :liam-E
K.P.L. and D.-G.W. contributed equally to this article equally.