Role For Pin1 Research Articles

Abstract PO-113: The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. PIN1 overexpression in cancer contributes to cancer cell-intrinsic phenotypes including cellular proliferation and migration. While its pro-tumor functions have generated interest in therapeutic targeting of PIN1 for cancer treatment, the effects of PIN1 inhibition on tumor-associated stromal phenotypes have not yet been studied. We assessed pancreatic cancer xenografts and genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice that were treated with small molecule PIN1 inhibitors or crossed into a full body PIN1 knockout (Pin1−/−), and found that PIN1 inhibition or loss decreased tumor growth and extended overall survival. To interrogate a direct role for PIN1 in the stroma, we orthotopically injected a KPC cell line into syngeneic Pin1+/+ or Pin1−/− hosts and found dramatic reduction of tumor cell growth in Pin1−/− hosts. Further analysis of the Pin1−/− tumor microenvironment revealed decreased expression of alpha-SMA, a marker of myofibroblastic cancer associated fibroblasts (myCAFs), as well as decreased ECM deposition and/or organization. Pancreatic stellate cells (PSCs) activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. We, therefore, interrogated the role of PIN1 in PSCs. We found that loss of PIN1 in PSCs inhibits TGF-beta-induced stellate cell activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive changes to chromatin accessibility are impaired in the absence of PIN1, and suggests that specific transcription factor families may play a role in the PIN1-dependent response to TGF-beta. Further analysis of PSCs or CAFs with PIN1 loss indicated that, at baseline, these cells express gene programs consistent with the recently described antigen presenting CAFs (apCAFs). Finally, in addition to changes in cellular state and plasticity, we found that loss of PIN1 alters PSC secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of VEGF, resulting in altered cancer cell and vascular phenotypes. This work establishes a role for PIN1 in regulating fibroblast function and suggests that targeting PIN1 in cancer will have a broad anti-tumor effect. Our ongoing work continues to use 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the precise mechanisms by which PIN1 controls fibroblast phenotypes and impact of these changes on tumor phenotypes and outcomes. Citation Format: Ellen M. Langer, Isabel A. English, Vidhi Shah, Kevin MacPherson, Kayleigh M. Kresse, Brittany L. Allen-Petersen, Colin J. Daniel, Mara H. Sherman, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-113.

Abstract LT012: The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. Due to its role in activation and stabilization of many oncogenes, we hypothesized that targeting PIN1 in pancreatic ductal adenocarcinoma (PDA) would slow tumor growth. We tested this hypothesis in vitro and in vivo with PIN1 inhibitors and/or genetic model systems. Pancreatic cancer cell lines knocked down for PIN1 or treated with PIN1 inhibitors showed decreased proliferation, invasion, and anchorage independent growth compared to control lines. Consistent with these in vitro results, treatment of pancreatic cancer xenografts or genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice with PIN1 inhibitors decreased tumor growth and extended overall survival. Similar results were seen in KPC mice that were crossed into a full body PIN1 knockout (PIN1−/−). Further analysis of KPC PIN1−/− tumors revealed not only reduced size of pancreatic tumors, but also decreased alpha-SMA expression and decreased ECM deposition in the stroma surrounding the tumors. PDA is characterized by a dense, desmoplastic tumor stroma that contributes to tumor growth, metastasis, and therapeutic resistance. Pancreatic stellate cells (PSCs) that are activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. To interrogate a direct role for PIN1 in the stroma, we first orthotopically injected a KPC cell line into syngeneic PIN1+/+ or PIN1−/− mice and found dramatic reduction of tumor cell growth in PIN1−/− hosts. Next, we analyzed PSCs in vitro and found that loss of PIN1 reduces their proliferation and alters their secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of SPINT1 and SPINT2, inhibitors of HGF activation. Conditioned media from control PSCs, but not from PSCs lacking PIN1 expression, activates the MET receptor on cancer cell lines, resulting in altered cancer cell phenotypes. In addition, we show that loss of PIN1 in PSCs inhibits TGF-beta induced stellate cells activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive chromatin changes are impaired in the absence of PIN1. Our ongoing work utilizes 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the mechanisms by which fibroblast phenotypes and the tumor-stromal crosstalk is impacted by PIN1. Citation Format: Ellen M. Langer, Isabel A. English, Kayleigh M. Kresse, Kevin MacPherson, Brittany L. Allen-Petersen, Colin J. Daniel, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT012.

Pin1 plays a key role in the response to treatment and clinical outcome in triple negative breast cancer.

Background:Triple negative breast cancer (TNBC) is the subset of breast cancer associated with the poorest outcome, and currently lacks targeted treatments. Standard of care (SoC) chemotherapy often consists of DNA damaging chemotherapies ± taxanes, with a range of responses observed. However, we currently lack biomarkers to predict this response and lack alternate treatment options.Methods:Pin1 expression was modulated in vitro and proliferation and treatment response was studied. Pin1 expression was analysed in patient samples and correlated with clinical outcome.Results:In this study, we have shown that the prolyl isomerase, Pin1, which is highly expressed in TNBC, plays a key role in pathogenesis of the disease. Knockdown of Pin1 in TNBC resulted in cell death while the opposite is seen in normal cells. We revealed for the first time that loss of Pin1 leads to increased sensitivity to Taxol but only in the absence of functional BRCA1. Conversely, loss of Pin1 results in decreased sensitivity to DNA-damaging agents independent of BRCA1 status. Analysis of Pin1 gene or IHC-based expression in over 200 TNBC patient samples revealed a novel role for Pin1 as a TNBC-specific biomarker, with high expression associated with improved outcome in the context of SoC chemotherapy. Preliminary data indicated this may be extended to other treatment options (e.g. Cisplatin/Parp Inhibitors) that are gaining traction for the treatment of TNBC.Conclusions:This study highlights the important role played by Pin1 in TNBC and highlights the context-dependent functions in modulating cell growth and response to treatment.

Invited commentary

Cellular senescence is an important contributor to the development of atherosclerosis. The mechanisms by which senescence promotes atherosclerosis, however, remain poorly understood. This important study by Lv et al demonstrates the causal association between reduced expression of peptidyl-prolyl cis/trans isomerase (Pin 1), increased vascular smooth muscle cell (VSMC) senescence, increased cellular senescence molecules p21 and p53, and reduced cell cycle-related cyclin-dependent kinases based on gain- and loss-of-function studies performed in vitro. In subsequent experiments, inhibition of Pin 1 was found to exacerbate experimental atherosclerosis. Thus, the authors postulate that Pin 1, likely acting upstream of the cellular senescence cascade, limits atherosclerosis by limiting VSMC senescence. An unexpected finding was that manipulation of the Pin 1 gene in vitro altered the expression of messenger RNA for p21, p53, p65, Gadd45, and cyclin-dependent kinases. These modulations are not attributable to variation in Pin 1 isomerase activity per se, leaving open the question of exactly which Pin 1-mediated actions suppress VSMC senescence. Senescent VSMCs express high levels of proatherogenic secretory molecules, which promote cell migration, extracellular matrix degradation, and the formation and activity of the inflammasome.1Gardner S.E. Humphry M. Bennett M.R. Clarke M.C. Senescent vascular smooth muscle cells drive inflammation through an interleukin-1alpha-dependent senescence-associated secretory phenotype.Arterioscl Thromb Vasc Biol. 2015; 35: 1963-1974Crossref PubMed Scopus (164) Google Scholar Further experiments should investigate how Pin 1 affects the VSMC senescence-associated secretory phenotype. In addition to senescent VSMCs, senescent foam cells are potent promoters of atherogenesis.2Childs B.G. Baker D.J. Wijshake T. Conover C.A. Campisi J. van Deursen J.M. Senescent intimal foam cells are deleterious at all stages of atherosclerosis.Science. 2016; 354: 472-477Crossref PubMed Scopus (605) Google Scholar This raises possibility that senescent foam cells may contribute to Pin 1 inhibition-mediated atherosclerosis. Thus, further experiments should be performed using VSMC- or myeloid cell-specific Pin 1-deficient mice to clarify the relative contributions of VSMC- and macrophage-derived Pin 1 to atherogenesis. The potential influence of Pin 1 on autophagy should also be investigated, given the causal association between defective autophagy and atherosclerosis.3Grootaert M.O. da Costa Martins P.A. Bitsch N. Pintelon I. De Meyer G.R. Martinet W. et al.Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis.Autophagy. 2015; 11: 2014-2032Crossref PubMed Scopus (178) Google Scholar Several polymorphisms in the gene encoding for Pin 1 are associated with a reduced risk for various cancers.4Xu H.R. Xu Z.F. Sun Y.L. Han J.J. Li Z.J. The -842G/C polymorphisms of PIN1 contributes to cancer risk: a meta-analysis of 10 case-control studies.PLoS One. 2013; 8: e71516Crossref PubMed Scopus (3) Google Scholar These or other known Pin 1 variants should be investigated to shed more light on the heritable susceptibility for atherosclerosis. Also, do hyperlipidemia and diabetes, two major atherosclerotic risk factors, influence Pin 1 expression? In a previous study,5Paneni F. Costantino S. Castello L. Battista R. Capretti G. Chiandotto S. et al.Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes.Eur Heart J. 2015; 36: 817-828Crossref PubMed Scopus (71) Google Scholar circulating monocytes harvested from diabetic patients demonstrate increased Pin 1 expression, and hyperglycemia has been found to upregulate expression in human endothelial cells in vitro. This study adds to the growing body of knowledge regarding the role of Pin 1 function in the regulation of atherosclerosis. Systemic augmentation of Pin 1 will isomerize phosphorylated serine/threonine-proline motif-containing proteins throughout the body, limiting clinical applicability due to undesirable off-target effects. And to date, no Pin 1 agonists or activators have been developed for clinical applications. Overcoming these obstacles will be necessary for the successful clinical translation of Pin 1-mediated therapeutic interventions in the treatment of atherosclerotic diseases. Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescenceJournal of Vascular SurgeryVol. 68Issue 3PreviewPin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. Full-Text PDF Open Archive

Dual Roles of Pin1 in Cancer Development and Progression.

Pin1 is a unique peptidyl-prolyl cis/trans isomerase (PPIase) that catalyzes the cis/trans isomerization of peptidyl-prolyl peptide bonds of its substrate proteins by binding to their specific phosphorylated Ser/Thr-Pro (pSer/Thr-Pro) motifs. This alters the conformation of target proteins and consequently affects their stability, intracellular localization, and/or biological functions. The abnormal overexpression of Pin1 is observed in some malignancies, which is associated with cancer cell proliferation, migration and invasion. However, a role for Pin1 as a putative tumor suppressor has recently been suggested. Systematic dissection of pro-oncogenic vs. tumor suppressive functions of Pin1 will be necessary.

Abstract 830: Pin1 is a novel target of withaferin A, a mammary cancer chemopreventative steroidal lactone from Withania somnifera

Abstract We have shown previously that naturally occurring steroidal lactone withaferin A (WA) inhibits breast cancer development in a transgenic mouse model. Mammary cancer chemoprevention by WA in vivo was associated with suppression of peptidyl-prolyl isomerase Pin1 protein. Pin1 is known to regulate diverse cellular processes including cell proliferation, cell cycle progression, apoptosis, and migration. Moreover, Pin1 is overexpressed in breast tumor and its expression is positively correlated with tumor grade. Thus, we studied functional significance of Pin1 downregulation by WA. Consistent with in vivo observations, Pin1 protein and mRNA expression was decreased upon WA treatment in human breast cancer cells irrespective of ER, HER-2 or p53 status. Overexpression of Pin1 partly reversed the growth inhibitory effect of WA in both MCF-7 (wild-type p53) and SUM159 (mutant p53) cells. However, WA-induced mitotic arrest and apoptosis were partly abrogated only in MCF-7 cells upon ectopic expression of Pin1 perhaps due to p53 stabilization. Molecular docking suggested covalent binding of WA with Cys113 of Pin1, which was confirmed by mass spectrometry. Overexpression of mutant (C113A) Pin1 augmented WA-mediated inhibition of cell survival as well as mitotic arrest and apoptosis in MCF-7 cells indicating that decreased catalytic activity of Pin1 is essential for growth inhibition by WA in breast cancer cells. Taken together, the present study suggests a critical role for Pin1 in WA-mediated growth inhibition of breast cancer. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Suman K. Samanta, Shivendra V. Singh. Pin1 is a novel target of withaferin A, a mammary cancer chemopreventative steroidal lactone from Withania somnifera. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 830.

B-cell activating factor-receptor specific activation of tumor necrosis factor receptor associated factor 6 and the phosphatidyl inositol 3-kinase pathway in lymphoma B cells

B-cell activating factor-receptor (BAFF-R) is the primary BAFF receptor that is responsible for promoting B-cell development and survival. Malignant B-cells exploit the BAFF/BAFF-R system, and high serum BAFF levels or genetic alterations in BAFF receptors have been found in B-cell cancers. BAFF signaling impacts pro-survival pathways. However, other than nuclear factor-κB2 (NF-κB2), little is known about the specific pathways activated by individual BAFF receptors. Using a novel BAFF-R expression model we have demonstrated that activation of BAFF-R, independent of transmembrane activator and cytophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), can induce phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β). Expression of an activated form of BAFF-R also enhanced a pro-survival gene expression pattern, including the novel BAFF-regulated gene Pin1, whose expression was phosphatidyl inositol 3-kinase (PI3K)-dependent. Additionally, we showed that TRAF6 is essential for mediating BAFF-R dependent activation of Akt. Together these data describe a novel role for TRAF6 in BAFF-R-specific activation of the PI3K pathway and provide evidence suggesting a new role for Pin1 in BAFF-R signaling.

Prolyl‐isomerase Pin1 controls normal and cancer stem cells of the breast

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers.

Hyperthermia Stress Activates Heat Shock Protein Expression via Propyl Isomerase 1 Regulation with Heat Shock Factor 1

Heat shock proteins (HSPs), which are members of the chaperone family of proteins, are essential factors for cellular responses to environmental stressors, such as hyperthermia, and are antiapoptotic. The transcription of HSPs is mainly controlled by heat shock transcription factor 1 (HSF1). In response to environmental stress, HSF1 forms a trimer, undergoes hyperphosphorylation, and is translocated to the nucleus. In this study, we show that upon heat shock treatment of cells, a WW domain-containing propyl-isomerase, PIN1, is able to colocalize to and associate with phospho-HSF1 at Ser(326) in the nucleus via its WW domain. This interaction is required for the DNA-binding activity of HSF1 and is consistent with the lower induction of HSPs in PIN1-deficient cells. This function of PIN1 is further demonstrated by in vivo refolding and survival assays, which have shown that PIN1-deficient cells are temperature sensitive and develop apoptosis upon exposure to an environmental challenge. Moreover, the reduced levels of HSPs in PIN1-deficient cells resulted in less efficient refolding of denatured proteins. Based on our results, we propose a novel role for PIN1 whereby it acts as a stress sensor regulating HSF1 activity in response to stress on multiple levels through the transcriptional activation of stress response elements in embryonic fibroblast cells, tumor cells, and neurons.

Pin1 Regulates the Dynamics of c-Myc DNA Binding To Facilitate Target Gene Regulation and Oncogenesis

The Myc oncoprotein is considered a master regulator of gene transcription by virtue of its ability to modulate the expression of a large percentage of all genes. However, mechanisms that direct Myc's recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl isomerase enhances recruitment of serine 62-phosphorylated Myc and its coactivators to select promoters during gene activation, followed by promoting Myc's release associated with its degradation. This facilitates Myc's activation of genes involved in cell growth and metabolism, resulting in enhanced proproliferative activity, even while controlling Myc levels. In cancer cells with impaired Myc degradation, Pin1 still enhances Myc DNA binding, although it no longer facilitates Myc degradation. Thus, we find that Pin1 and Myc are cooverexpressed in cancer, and this drives a gene expression pattern that we show is enriched in poor-outcome breast cancer subtypes. This study provides new insight into mechanisms regulating Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in the regulation of transcription factors, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and Myc.

Abstract 771: Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis.

Abstract The c-Myc oncoprotein is generally considered to be a master regulator of gene transcription by virtue of its ability to activate or repress the expression of a large percentage of all genes. However, mechanisms that direct c-Myc's recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl-isomerase regulates c-Myc transcriptional activity by altering c-Myc's association with target gene promoters. Specifically, we show that Pin1 enhances the recruitment of Serine62-phosphorylated c-Myc along with its co-activators to selected target gene promoters during gene activation, followed by promoting c-Myc's release associated with its degradation, resulting in cyclic c-Myc DNA binding. This facilitates c-Myc's activation of target genes associated with an increase in elongating RNA Polymerase II in the gene body. Genes impacted by this mechanism are involved in cell growth and metabolism, resulting in enhanced pro-proliferative activity, even while controlling c-Myc expression. In cancer cells with impaired c-Myc degradation, Pin1 still regulates cyclic c-Myc DNA binding, even though it no longer enhances c-Myc degradation. This allows for simultaneous high expression of Pin1 and c-Myc in cancer, which can drive a gene expression pattern that we show is enriched in poor outcome subtypes of breast cancer. This study provides new insight into mechanisms regulating c-Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in transcription factor regulation, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and c-Myc. Citation Format: Amy Farrell, Carl Pelz, Xiaoyan Wang, Colin Daniel, Yulong Su, Mahnaz Janghorban, Soren Impey, Rosalie C. Sears. Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2013-771

Abstract 49: Prolyl Isomerase Pin1 Regulates Cardiac Hypertrophy via Controlling Phosphorylation of Akt and MEK

Rationale: Kinases and phosphatases regulate crucial aspects of growth and survival through phosphorylation and dephosphorylation of target substrates. Processes of cardiac hypertrophy, myocardial infarction, and heart failure are dictated in part by which kinases or phosphatases are involved and also by the intensity and duration of specific enzymatic activities. While research has identified numerous critical regulatory kinases and phosphatases in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the non-myocyte context, control of folding, activity, stability, and subcellular localization of proteins responsible for growth and survival is mediated by the prolyl isomerase Pin1. Objective: To establish the role of Pin1 in the heart. Method and Results: Initial characterization of myocardial Pin1 involved assessment of expression and localization during postnatal development or pathological challenge. Pin1 protein level was decreased and the location of Pin1 was changed from nucleus to cytoplasm with increasing age. Next, Pin1 protein expression and localization were assessed in the pathological challenged heart. Pin1 protein expression increases with pressure overload and ischemia, particularly in perivascular areas and in border zone myocytes, respectively. To determine the role of Pin1 on cardiac hypertrophy, siRNA to Pin1 (siPin1) was applied to neonatal rat cardiomyocytes. Western blot analysis showed that siPin1 decreased phosphorylation of Akt, and immunohistochemical analysis showed that siPin1 reduced cardiomyocyte size in response to high serum. siPin1 also decreased phosphorylation of MEK and reduced cardiomyocyte size in response to phenylephrine treatment. Furthermore, cardiac hypertrophy induced by transaortic constriction was ameliorated in Pin1 knockout mice, compared with littermate wild type mice. Conclusion: Expression and location of Pin1 during development and in response to pathologic challenge point to an important role for Pin1 in adaptation to myocardial growth or stress. Collective evidence indicates that Pin1 controls cardiac hypertrophy at least in part via regulating phosphorylation of Akt and MEK.

The Prolyl Isomerase Pin1 Modulates Development of CD8+ cDC in Mice

BackgroundPin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses.Methodology/Principal FindingsWhen challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life.Conclusions/SignificanceWe have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

Abstract A31: Effects of Pin1 inhibition on tumorigenicity of glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor, accounting for 20% of all intracranial tumors. Current approaches in pursuit of treatment of GBM are not efficient in terms of increasing patient survivability or preventing recurrence after traditional treatments. Hunt for key molecules that might turn into drug targets have been rigorously continuing for GBM as well as other various types of tumors. Pin1, which was shown to be broadly overexpressed in various types of tumors, bears the potential to be one of the key molecular players in tumorigenesis. Molecular mechanisms underlying development and growth of the GBM, which is not completely curable by the means of chemotherapy, radiotherapy, and surgery, are not yet clearly identified. For the fact that the rate of treatment success of the agents in use against GBM is lower than acceptations, investigations for potential inhibitory target molecules for GBM development are increasing. Pin1 (peptidyl-prolyl cis/trans isomerase) is one of the molecules, recently identified and trying to be clarified during the search for potential target molecules on tumorigenicity. When compared with the health cells level of the Pin1 protein in many tumor cells like prostate, lung, breast and brain tumors is found to be extremely high. Recent studies aiming to identify Pin1's role on tumorigenicity have shown that increase in level of Pin1 in cells also led in increase of VEGF (Vascular Endothelial Growth Factor) as well. Our study aims to reveal the possible role for Pin1 as a therapeutic target and also inhibit the angiogenesis mechanism, which is common for tumors to develop, through inhibition of Pin1 U87 MG Glioblastoma cell line was used and Pin1 inhibition was performed via juglone, a Pin1 specific inhibitor molecule, and Pin1 siRNAs. Effects on Pin1 inhibition on tumorigenicity was assessed by MTT proliferation assay, wound healing assay to observe the migration characteristics, confocal microscopy technique to define morphological changes and apoptosis, and western blotting to define if angiogenesis related proteins are expressed. It is observed that Pin1 inhibition has affected and diminished the migration ability of U87 MG Glioblastoma cells. Changes on cell morphology and activation of apoptosis were also observed with reduced expression levels of angiogenesis related genes. We believe that, this study has a potential to reveal the effects of Pin1 inhibition as a therapeutic approach and will contribute to literature and clinical care in the future. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A31.

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism

Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3' UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP-PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1-/- mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.

The Peptidyl-Prolyl Isomerase Pin1 Regulates Cytokinesis through Cep55

Failure of cytokinesis results in tetraploidy and can increase the genomic instability frequently observed in cancer. The peptidyl-prolyl isomerase Pin1, which is deregulated in many tumors, regulates several processes, including cell cycle progression. Here, we show a novel role for Pin1 in cytokinesis. Pin1 knockout mouse embryonic fibroblasts show a cytokinesis delay, and depletion of Pin1 from HeLa cells also causes a cytokinesis defect. Furthermore, we provide evidence that Pin1 localizes to the midbody ring and regulates the final stages of cytokinesis by binding to centrosome protein 55 kDa (Cep55), an essential component of this ring. This interaction induces Polo-like kinase 1-mediated phosphorylation of Cep55, which is critical for the function of Cep55 during cytokinesis. Importantly, Pin1 knockdown does not enhance the cytokinesis defect in Cep55-depleted cells, indicating that Pin1 and Cep55 act in the same pathway. These data are the first evidence that Pin1 regulates cytokinesis and may provide a mechanistic explanation as to how pathologic levels of Pin1 can stimulate tumorigenesis.

A role for Pin1 in mammalian germ cell development and spermatogenesis

The peptidyl-prolyl isomerase Pin1 is proposed to have diverse functions in many vital aspects of the cell. Despite the multitude of proteins targeted by Pin1 and the proposed regulatory role it plays in critical cellular functions, Pin1 is an essential gene in some eukaryotic organisms, but is dispensable in metazoans. In two genetic models, Candida albicans and Drosophila melanogaster, Pin1 participates in distinct developmental processes regulated by the MAPK pathway. Pin1-deficient mice exhibit decreased primordial germ cell proliferation during embryonic development, along with several degenerative or proliferative defects in the adult testis, retina, mammary gland, and brain. The combination of primordial germ cell deficit and spermatogonial depletion contributes to severe fertility defects in Pin1-null mice. Since growth factor activated MAPK pathways are vital to germ cell proliferation and differentiation, a role for Pin1 in mammalian germ cell development and spermatogenesis is discussed in the context of the Ras/MEK/MAPK pathway.

Interactions between Protein Kinase CK2 and Pin1: EVIDENCE FOR PHOSPHORYLATION-DEPENDENT INTERACTIONS

The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha' subunits together with two regulatory CK2 beta subunits. Our results indicate that Pin1 can interact with CK2 complexes that contain CK2 alpha. Furthermore, Pin1 can interact directly with the C-terminal domain of CK2 alpha that contains residues that are phosphorylated in vitro by p34(Cdc2) and in mitotic cells. Substitution of the phosphorylation sites of CK2 alpha with alanines resulted in decreased interactions between Pin1 and CK2. The other catalytic isoform of CK2, designated CK2 alpha', is not phosphorylated in mitotic cells and does not interact with Pin1, but a chimeric protein consisting of CK2 alpha' with the C terminus of CK2 alpha was phosphorylated in mitotic cells and interacts with Pin1, further implicating the phosphorylation sites in the interaction. In vitro, Pin1 inhibits the phosphorylation of Thr-1342 on human topoisomerase II alpha by CK2. Topoisomerase II alpha also interacts with Pin1 suggesting that the effect of Pin1 on the phosphorylation of Thr-1342 could result from its interactions with CK2 and/or topoisomerase II alpha. As compared with wild-type Pin1, isomerase-deficient and WW domain-deficient mutants of Pin1 are impaired in their ability to interact with CK2 and to inhibit the CK2-catalyzed phosphorylation of topoisomerase II alpha. Collectively, these results indicate that Pin1 and CK2 alpha interact and suggest a possible role for Pin1 in the regulation of topoisomerase II alpha. Furthermore, these results provide new insights into the functional role of the mitotic phosphorylation of CK2 and provide a new mechanism for selectively regulating the ability of CK2 to phosphorylate one of its mitotic targets.

Pin1 modulates the dephosphorylation of the RNA polymerase II C-terminal domain by yeast Fcp1

The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr–Pro) is an important signaling mechanism in the cell. The pSer/Thr–Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl–prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1’s PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser5–Pro motif, but not on Ser2–Pro in yeast, which can be explained with Pin1’s substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation.