Role Of Pin1 Research Articles

Prolyl isomerase Pin1 in skeletal muscles contributes to systemic energy metabolism and exercise capacity through regulating SERCA activity

The skeletal muscle is a pivotal organ involved in the regulation of both energy metabolism and exercise capacity. There is no doubt that exercise contributes to a healthy life through the consumption of excessive energy or the release of myokines. Skeletal muscles exhibit insulin sensitivity and can rapidly uptake blood glucose. In addition, they can undergo non-shivering thermogenesis through actions of both the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and small peptide, sarcolipin, resulting in systemic energy metabolism. Accordingly, the maintenance of skeletal muscles is important for both metabolism and exercise. Prolyl isomerase Pin1 is an enzyme that converts the cis-trans form of proline residues and controls substrate function. We have previously reported that Pin1 plays important roles in insulin release, thermogenesis, and lipolysis. However, the roles of Pin1 in skeletal muscles remains unknown. To clarify this issue, we generated skeletal muscle-specific Pin1 knockout mice. Pin1 deficiency had no effects on muscle weights, morphology and ratio of fiber types. However, they showed exacerbated obesity or insulin resistance when fed with a high-fat diet. They also showed a lower ability to exercise than wild type mice did. We also found that Pin1 interacted with SERCA and elevated its activity, resulting in the upregulation of oxygen consumption. Overall, our study reveals that Pin1 in skeletal muscles contributes to both systemic energy metabolism and exercise capacity.

Prolyl isomerase Pin1 promotes autophagy and cancer cell viability through activating FoxO3 signalling.

Pin1-directed prolyl isomerization is a central common oncogenic mechanism to drive tumorigenic processes. However, the role of Pin1 in cellular autophagy is still poorly understood. Here we report that pharmacological inhibition of Pin1 decreased the formation of autophagosome/autolysosomes upon nutrient starvation. Inhibition of Pin1 reduced, whereas forced expression of Pin1 increased, the level of LC3 and viability of U2OS and PANC-1 cells. Pin1 could augment the accumulation of LC3 upon chloroquine treatment, while chloroquine also disturbed its function on cell viability. RNA-Seq and qPCR identified altered autophagic pathway upon Pin1 silencing. Mechanistically, FoxO3 was identified critical for Pin1-mediated autophagy. Knockdown of FoxO3 could rescue the changes of LC3 level and cellular viability caused by Pin1 overexpression. In xenograft mouse model, Pin1 reduced the sensitivity of PANC-1 to chloroquine while FoxO3 silencing could inhibit Pin1's function. Moreover, Pin1 could bind FoxO3 via its pS284-P motif, reduce its phosphorylation at T32, facilitate its nuclear retention, and therefore increased its transcriptional activity. S284A mutation of FoxO3 interfered with its T32 phosphorylation, reduced its nuclear localization and disrupted its function to support cell viability upon nutrient starvation. Furthermore, the protein level of Pin1 positively correlated with FoxO3 nuclear localization and LC3 level in pancreatic adenocarcinoma and osteosarcoma samples. Together, this study highlights an important role for Pin1-FoxO3 axis in regulating autophagy and cancer cell viability. Intervening in the Pin1-FoxO3 interaction would serve as an effective therapeutic strategy and the pS284-P motif of FoxO3 provides a potential target for drug design.

Role of PIN1 in human pathology: Cellular regulation, pathogenesis and therapeutic implications (Review)

Role of PIN1 in human pathology: Cellular regulation, pathogenesis and therapeutic implications (Review)

A bibliometric analysis of PIN1 and cell death.

Background: Regulation of cell death plays a key role in numerous diseases. As a proline isomerase, prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is important for the regulation of signaling pathways. An in-depth understanding of how Pin1 participates in the process of cell death, which affects the occurrence and development of diseases, will aid in the discovery of new disease mechanisms and therapeutic methods. Thus, the purpose of our study was to discover the research trends and hotspots of Pin1 and cell death through bibliometric analyses and to provide insights for understanding the future development of basic research and treatment of diseases. Methods: Documents were extracted from the Web of Science Core Collection on 7 May 2022. We selected articles and reviews published in English from 2000 to 2021, and visual and statistical analyses of countries, institutions, authors, references and keywords were performed using VOSviewer 1.6.18 and CiteSpace 5.8. Results: A total of 395 articles and reviews were selected. Since 2001, the number of articles on Pin1 and cell death has increased annually. Publications come from 43 countries, with the US having the most publications and citations. We identified 510 authors, with Giannino Del Sal having the most articles and Paola Zacchi having the most co-citations. The Journal of Biological Chemistry is the most researched journal, and Nature and its subjournals are the most cited journals. Apoptosis, phosphorylation, and breast cancer were the three most common keywords. Conclusion: The number of documents showed an increasing trend from 2001 to 2014. Stagnant growth after 2014 may be related to the absence of new research hotspots. Cooperative links between core institutions need to be strengthened, and the institution with the highest citation count in recent years is Fujian Medical University in China. The role of Pin1 in cell death requires further research to discover new research hotspots. Before breakthroughs in molecular mechanism or signaling pathway research, future research will focus more on the treatment of diseases represented by Pin1 inhibitors.

The role of peptidyl-prolyl isomerase Pin1 in neuronal signaling in epilepsy.

Epilepsy is a common symptom of many neurological disorders and can lead to neuronal damage that plays a major role in seizure-related disability. The peptidyl-prolyl isomerase Pin1 has wide-ranging influences on the occurrence and development of neurological diseases. It has also been suggested that Pin1 acts on epileptic inhibition, and the molecular mechanism has recently been reported. In this review, we primarily focus on research concerning the mechanisms and functions of Pin1 in neurons. In addition, we highlight the significance and potential applications of Pin1 in neuronal diseases, especially epilepsy. We also discuss the molecular mechanisms by which Pin1 controls synapses, ion channels and neuronal signaling pathways to modulate epileptic susceptibility. Since neurotransmitters and some neuronal signaling pathways, such as Notch1 and PI3K/Akt, are vital to the nervous system, the role of Pin1 in epilepsy is discussed in the context of the CaMKII-AMPA receptor axis, PSD-95-NMDA receptor axis, NL2/gephyrin-GABA receptor signaling, and Notch1 and PI3K/Akt pathways. The effect of Pin1 on the progression of epilepsy in animal models is discussed as well. This information will lead to a better understanding of Pin1 signaling pathways in epilepsy and may facilitate development of new therapeutic strategies.

The kingdom of the prolyl-isomerase Pin1: The structural and functional convergence and divergence of Pin1

More than 20 years since its discovery, our understanding of Pin1 function in various diseases continues to improve. Pin1 plays a crucial role in pathogenesis and has been implicated in metabolic disorders, cardiovascular diseases, inflammatory diseases, viral infection, cancer and neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease. In particular, the role of Pin1 in neurodegenerative diseases and cancer has been extensively studied. Our understanding of Pin1 in cancer also led to the development of cancer therapeutic drugs targeting Pin1, with some currently in clinical trial phases. However, identifying a Pin1-specific drug with good cancer therapeutic effect remains elusive, thus leading to the continued efforts in Pin1 research. The importance of Pin1 is highlighted by the presence of Pin1 orthologs across various species: from vertebrates to invertebrates and Kingdom Animalia to Plantae. Among these Pin1 orthologs, their sequence and structural similarity demonstrate the presence of conservation. Moreover, their similar functionality between species further highlights the conservancy of Pin1. As researchers continue to unlock the mysteries of Pin1 in various diseases, using different Pin1 models might shed light on how to better target Pin1 for disease therapeutics. This review aims to highlight the various Pin1 orthologs in numerous species and their divergent functional roles. We will examine their sequence and structural similarities and discuss their functional similarities and uniqueness to demonstrate the interconnectivity of Pin1 orthologs in multiple diseases.

Pin1 aggravates renal injury induced by ischemia and reperfusion in rats via Nrf2/HO-1 mediated endoplasmic reticulum stress.

ABSTRACTPurpose:To investigate the role of peptidyl-prolyl cis/trans isomerase 1 (Pin1) on renal ischemia-reperfusion (I/R) injury and underlying mechanism.Methods:By establishing the in vitro and in vivo models of renal I/R, the role of Pin1 was explored by using molecular assays.Results:In renal I/R, endogenous Pin1 level was up-regulated in I/R-impaired kidney. Suppression of Pin1 with juglone afforded protection against I/R-mediated kidney dysfunction, and reduced I/R-induced endoplasmic reticulum (ER) stress in vivo. Consistent with the in vivo results, repression of Pin1 with juglone or gene knockdown with si-Pin1 conferred cytoprotection and restricted hypoxia/reoxygenation (H/R)-driven ER stress in HK-2 cells. Simultaneously, further study uncovered that Nrf-2/HO-1 signals was the association between Pin1 and ER stress in response to renal I/R. In addition, Nrf-2/HO-1 signal pathway was inactivated after kidney exposed to I/R, as indicated by the down-regulation of Nrf-2/HO-1 levels. Furthermore, inhibition of Pin1 remarkably rescued the inactivation ofNrf-2/HO-1.Conclusions:Pin1 modulated I/R-mediated kidney injury in ER stress manner dependent on Nrf2-HO-1 pathway in I/R injury.

Pin1 as Molecular Switch in Vascular Endothelium: Notes on Its Putative Role in Age-Associated Vascular Diseases.

By controlling the change of the backbones of several cellular substrates, the peptidyl-prolyl cis-trans isomerase Pin1 acts as key fine-tuner and amplifier of multiple signaling pathways, thereby inducing several biological consequences, both in physiological and pathological conditions. Data from the literature indicate a prominent role of Pin1 in the regulating of vascular homeostasis. In this review, we will critically dissect Pin1’s role as conformational switch regulating the homeostasis of vascular endothelium, by specifically modulating nitric oxide (NO) bioavailability. In this regard, Pin1 has been reported to directly control NO production by interacting with bovine endothelial nitric oxide synthase (eNOS) at Ser116-Pro117 (human equivalent is Ser114-Pro115) in a phosphorylation-dependent manner, regulating its catalytic activity, as well as by regulating other intracellular players, such as VEGF and TGF-β, thereby impinging upon NO release. Furthermore, since Pin1 has been found to act as a critical driver of vascular cell proliferation, apoptosis, and inflammation, with implication in many vascular diseases (e.g., diabetes, atherosclerosis, hypertension, and cardiac hypertrophy), evidence indicating that Pin1 may serve a pivotal role in vascular endothelium will be discussed. Understanding the role of Pin1 in vascular homeostasis is crucial in terms of finding a new possible therapeutic player and target in vascular pathologies, including those affecting the elderly (such as small and large vessel diseases and vascular dementia) or those promoting the full expression of neurodegenerative dementing diseases.

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.

Cobalt induces neurodegenerative damages through Pin1 inactivation in mice and human neuroglioma cells

Cobalt is a hazardous material that has harmful effects on neurotoxicity. Excessive exposure to cobalt or inactivation of the unique proline isomerase Pin1 contributes to age-dependent neurodegeneration. However, nothing is known about the role of Pin1 in cobalt-induced neurodegeneration. Here we find that out of several hazardous materials, only cobalt dose-dependently decreased Pin1 expression and alterations in its substrates, including cis and trans phosphorylated Tau in human neuronal cells, concomitant with neurotoxicity. Cobalt-induced neurotoxicity was aggravated by Pin1 genetic or chemical inhibition, but rescued by Pin1 upregulation. Furthermore, less than 4μg/l of blood cobalt induced dose- and age-dependent Pin1 downregulation in murine brains, ensuing neurodegenerative changes. These defects were corroborated by changes in Pin1 substrates, including cis and trans phosphorylated Tau, amyloid precursor protein, β amyloid and GSK3β. Moreover, blood Pin1 was downregulated in human hip replacement patients with median blood cobalt level of 2.514μg/l, which is significantly less than the safety threshold of 10μg/l, suggesting an early role Pin1 played in neurodegenerative damages. Thus, Pin1 inactivation by cobalt contributes to age-dependent neurodegeneration, revealing that cobalt is a hazardous material triggering AD-like neurodegenerative damages.

Abstract NT-106: PIN1: A PROMISING TARGET FOR PLATINUM-RESISTANT HIGH GRADE SEROUS OVARIAN CANCER

Abstract Epithelial ovarian cancer is the leading cause of death in gynaecological cancers in developed countries and the fifth most common cause of cancer mortality in women. High-grade serous epithelial ovarian cancer (HGS-EOC) is derived from the surface of the ovary and/or the distal fallopian tube: the relative contribution to the EOC remains to be determined. Currently, debulking surgery and chemotherapy are the standard therapy in ovarian cancer treatment. Evidence from different clinical trials established the paclitaxel and carboplatin combination regimen as the first-line chemotherapeutic treatment, although resistance occurs. Pin1, a peptidyl prolyl cis-trans isomerase, which controls different oncogenes and tumor suppressor genes, is overexpressed in about fifty percent of HGS-EOC suggesting that it may be a potential therapeutic target. Although many efforts have been dedicated to the development of Pin1 inhibitors, up to now a few are effective on cancer cells or in vivo mouse models. Our group recently published the first liposomal formulation of a potent and specific Pin1 inhibitor, which successfully constrains tumor growth in a mouse model of ovarian cancer. We demonstrated the involvement of Pin1 in HGS-EOC showing that it is overexpressed in ovarian cancer tissue samples and when genetically or chemically inhibited, promotes ovarian cancer cell death in vitro and in vivo (PMID: 29746956). Spheroids derived from SKOV3 cell line were utilized to better reproduce the spreading of cancer cells from ascitic fluids to secondary sites. After Pin1 chemical inhibition, the cells fail to form spheroids or spheroid growth and migration. Since platinum-resistance occurs in almost all HGS-EOC patients, we investigated the role of Pin1 as effective advanced therapy in 2D and 3D in vitro systems. In literature, it is reported that the genetic inhibition of Pin1 synergizes with cisplatin in cervical cancer. Our preliminary data showed that chemical or shRNAs inhibition of Pin1 in HGS- EOC cell lines sensitize the cells to carboplatin. To investigate which molecular pathways are involved, different key cancer proteins were examined. Pin1 affects PI3K/Akt pathway, by decreasing the expression of activated Akt protein, a key regulator of cancer cell proliferation, migration and survival. In conclusion, we propose Pin1 as promising targeted therapy of HGS-EOC patients with platinum-resistance. Citation Format: Concetta Russo Spena, Lucia De Stefano, Barbara Salis, Carlotta Granchi, Maguie El Boustani, Stefano Palazzolo, Nayla Mouawad, Tiziano Tuccinardi, Vincenzo Canzonieri, Isabella Caligiuri, Flavio Rizzolio. PIN1: A PROMISING TARGET FOR PLATINUM-RESISTANT HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-106.

Roles of Pin1 as a Key Molecule for EMT Induction by Activation of STAT3 and NF-κB in Human Gallbladder Cancer.

Despite developments in multidisciplinary treatment, the prognosis for advanced gallbladder cancer (GBC) still is poor because of its rapid progression. Epithelial-mesenchymal transition (EMT) plays a central role in promoting tumor invasion and metastasis in malignancies thorough signal transducer and activator of transcription-3 (STAT3) and nuclear factor κB (NF-κB) activation. Whereas Pin1 mediates STAT3 and NF-κB activation, the involvement of Pin1 in GBC progression is unclear. Factors regulating Pin1-related STAT3 and NF-κB activation were evaluated using surgical specimens collected from 76 GBC patients, GBC cells, and orthotopic GBC xenograft mice. In the patients with GBC, high Pin1 expression in GBC was associated with aggressive tumor invasion and increased tumor metastasis, and was an independent factor for a poor prognosis. Pin1 expression was correlated with phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276), thereby activating STAT3 and NF-κB in GBC. Pin1-mediated STAT3 and NF-κB activation induced EMT in GBC. When Pin1 knockdown was performed in GBC cells, the phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276) was inhibited, and STAT3 and NF-κB activation was suppressed. Inactivation of STAT3 and NF-κB in Pin1-depleted cells decreased snail and zeb-2 expression, thereby reducing the rate of mesenchymal-like cells, suggesting that EMT was inhibited in GBC cells. PiB, a Pin1-specific inhibitor, inhibited EMT and reduced tumor cell invasion by inactivating STAT3 and NF-κB in vitro. Moreover, PiB treatment inhibited lymph node metastasis and intrahepatic metastasis in orthotopic GBC xenograft tumor in vivo. Pin1 accelerates GBC invasion and metastasis by activating STAT3 and NF-κB. Therefore, Pin1 inhibition by PiB is an excellent therapy for GBC by safely inhibiting its metastasis.

The Multiple Roles of Peptidyl Prolyl Isomerases in Brain Cancer.

Peptidyl prolyl isomerases (PPIases) are broadly expressed enzymes that accelerate the cis-trans isomerization of proline peptide bonds. The most extensively studied PPIase family member is protein interacting with never in mitosis A1 (PIN1), which isomerizes phosphorylated serine/threonine–proline bonds. By catalyzing this specific cis-trans isomerization, PIN1 can alter the structure of its target proteins and modulate their activities in a number of different ways. Many proteins are targets of proline-directed phosphorylation and thus PIN1-mediated isomerization of proline bonds represents an important step in the regulation of a variety of cellular mechanisms. Numerous other proteins in addition to PIN1 are endowed with PPIase activity. These include other members of the parvulin family to which PIN1 belongs, such as PIN4, as well as several cyclophilins and FK506-binding proteins. Unlike PIN1, however, these other PPIases do not isomerize phosphorylated serine/threonine–proline bonds and have different substrate specificities. PIN1 and other PPIases are overexpressed in many types of cancer and have been implicated in various oncogenic processes. This review will discuss studies providing evidence for multiple roles of PIN1 and other PPIases in glioblastoma and medulloblastoma, the most frequent adult and pediatric primary brain tumors.

A3081 VDR agonist prevents diabetic endothelial dysfunction through inhibition of prolyl isomerase-1 mediated mitochondrial oxidative stress and inflammation

Objectives: Upregulation of prolyl isomerase-1 (Pin1) protein expression and activity was associated with the pathogenesis of diabetic vasculopathy through induction of endothelial oxidative stress and inflammation. Moreover, VDR agonist protects against high glucose-induced endothelial apoptosis through inhibition of oxidative stress. We aimed to explore the effects of VDR agonist on diabetes-associated endothelial dysfunction and the role of Pin1 in this process. Methods: Streptozocin-induced diabetic mice were treated with vehicle, VDR agonist or Pin1 inhibitor, Juglone for eight weeks. In parallel, human umbilical vein endothelial cells (HUVECs) exposed to high glucose condition were treated with 1,25-dihydroxyvitamin D3, and Juglone or vehicle for 72 hours. Organ chamber experiments were performed to assess endothelium-dependent relaxation to acetylcholine. Circulatory levels of Pin1, SOD, MDA, IL-1β, IL-6, and NO in diabetic mice, Pin1 protein expression and activity, subcellular distribution of p66Shc and NF-kB p65 in high glucose-cultured HUVECs were determined. Results: VDR agonist and Juglone significantly improved diabetes-associated endothelial dysfunction and reduced high glucose-induced endothelial apoptosis. Mechanistically, the circulatory levels of SOD and NO were increased compared with those in vehicle-treated diabetic mice. Additionally, Pin1 protein expression and activity, p66Shc mitochondrial translocation, and NF-kB p65 in high glucose-cultured HUVECs were also inhibited by VDR agonist and Juglone. Knockdown of VDR abolished the inhibitory effects of VDR agonist on high glucose-induced upregulation of Pin1 protein expression and activity. Conclusion: VDR agonist prevents diabetic endothelial dysfunction through inhibition of Pin1-mediated mitochondrial oxidative stress and inflammation.

Pin1 inhibition exerts potent activity against acute myeloid leukemia through blocking multiple cancer-driving pathways

BackgroundThe increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia.MethodsThe mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models.ResultsFirst, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models.ConclusionsWe demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML.

Abstract A20: Pin1 as a regulator of multiple targets of cell growth in lung cancer

Abstract Peptidylprolyl isomerase 1 (Pin1), a peptidyl-prolyl cis/trans isomerase, is overexpressed in many different cancer types and has been shown to serve as a poor prognostic factor in the lung. Furthermore, it has been indicated that overexpression of Pin1 correlates with increased expression of both p53 and MDM2 in human adenocarcinoma. However, the exact molecular mechanism by which Pin1 increases tumor cell proliferation is still unknown. Here, we show that Pin1 increases the occurrence of adenocarcinoma in a mouse model. Moreover, we assessed the hypothesis that Pin1 increases lung tumorigenesis by altering the function of alternative cell cycle regulators, perhaps affecting their phosphorylation status. Recently, several genetically engineered mouse models of lung adenocarcinoma have been developed to mimic the genetic and phenotypic features of the human disease, including activating mutations in K-RAS and loss-of-function mutations in p53. To fully understand the role of Pin1 in lung cancer, we developed a mouse model of Pin1 overexpression in the context of mutant K-RAS and p53 loss of function in immunocompetent mice. This model was obtained by an intratracheal delivery of lentiviral Cre-recombinase, which activates the expression of a mutant oncogene K-RAS (G12D) and a truncated p53, combined with ectopic overexpression of Pin1. Changes in protein expression and cellular localization of the proliferative index Ki67, RB1, RB2, and cdk2, upon Pin1 ectopic overexpression, were evaluated in sections of paraffin-embedded lung tumor tissue from our conditional mouse model at different time points, and using an immunohistochemical (IHC) approach. To evaluate the effects of Pin1 on lung tumor cell proliferation specific for cell cycle regulatory pathways, we also performed Western blot (WB) analysis using frozen tissue collected from the same conditional mouse model. The histologic classification of each samples was made on H&E-stained sections by A.K.S., a veterinary pathologist with extensive experience in murine lung pathology. Our results confirmed that overexpression of Pin1 correlates with increased expression of both p53 and MDM2, and more importantly indicated that Pin1 raises the occurrence of adenocarcinoma in vivo. Specifically, we found that Pin1 increases lung tumorigenesis by altering the phosphorylation status of alternative cell cycle regulatory pathways irrespective of the p53-cell cycle regulatory system. In conclusion, in our model the overexpression of Pin1 contributed to a higher incidence of lung adenocarcinoma, suggesting Pin1 as having a promoting effect toward carcinogenesis. Lung cancer continues to be one of the leading causes of cancer deaths in the US and, while surgical resection offers the best treatment option, oftentimes the cancer is not detected early enough for surgery to be effective. For this reason, identifying molecular targets centered on the early stages of lung cancer development that will aid in the advancement of treatments is of the utmost importance. We believe that Pin1, and its related targets, offer a unique therapeutic option to alter the cancer cell cycle machinery. Citation Format: Silvia Boffo, Barbara D'Angelo, Andres J. Klein-Szanto, Marcella Macaluso, Antonio Giordano. Pin1 as a regulator of multiple targets of cell growth in lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A20.

VDR Agonist Prevents Diabetic Endothelial Dysfunction through Inhibition of Prolyl Isomerase-1-Mediated Mitochondrial Oxidative Stress and Inflammation.

Background and aim Upregulation of prolyl isomerase-1 (Pin1) protein expression and activity was associated with the pathogenesis of diabetic vasculopathy through induction of endothelial oxidative stress and inflammation. Moreover, VDR agonist protects against high glucose-induced endothelial apoptosis through the inhibition of oxidative stress. We aimed to explore the effects of the VDR agonist on diabetes-associated endothelial dysfunction and the role of Pin1 in this process. Methods Streptozocin-induced diabetic mice were randomly treated with vehicle, VDR agonist (10 μg/kg/d, i.g., twice a week), or Pin1 inhibitor, Juglone (1 mg/kg/d, i.p., every other day), for eight weeks. In parallel, human umbilical vein endothelial cells (HUVECs) exposed to high-glucose condition were treated with 1,25-dihydroxyvitamin D3 and Juglone or vehicle for 72 hours. Organ chamber experiments were performed to assess endothelium-dependent relaxation to acetylcholine. Circulatory levels of Pin1, SOD, MDA, IL-1β, IL-6, and NO in diabetic mice, Pin1 protein expression and activity, subcellular distribution of p66Shc, and NF-κB p65 in high glucose-cultured HUVECs were determined. Results Both VDR agonist and Juglone significantly improved diabetes-associated endothelial dysfunction and reduced high glucose-induced endothelial apoptosis. Mechanistically, the circulatory levels of SOD and NO were increased compared with those of vehicle-treated diabetic mice. Additionally, Pin1 protein expression and activity, p66Shc mitochondrial translocation, and NF-κB p65 in high glucose-cultured HUVECs were also inhibited by VDR agonist and Juglone. Knockdown of VDR abolished the inhibitory effects of VDR agonist on high glucose-induced upregulation of Pin1 protein expression and activity. Conclusions VDR agonist prevents diabetic endothelial dysfunction through inhibition of Pin1-mediated mitochondrial oxidative stress and inflammation.

Pin1 facilitates isoproterenol‑induced cardiac fibrosis and collagen deposition by promoting oxidative stress and activating the MEK1/2‑ERK1/2 signal transduction pathway in rats.

Peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) is a member of a large superfamily of phosphorylation-dependent peptidyl-prolyl cis/trans isomerases, which not only regulates multiple targets at various stages of cellular processes, but is also involved in the pathogenesis of several diseases, including microbial infection, cancer, asthma and Alzheimer's disease. However, the role of Pin1 in cardiac fibrosis remains to be fully elucidated. The present study investigated the potential mechanism of Pin1 in isoprenaline (ISO)-induced myocardial fibrosis in rats. The rats were randomly divided into three groups. Echocardiography was used to evaluate changes in the size, shape and function of the heart, and histological staining was performed to visualize inflammatory cell infiltration and fibrosis. Reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and Picrosirius red staining were used to differentiate collagen subtypes. Additionally, cardiac-specific phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular-signal regulated protein kinase 1/2 (ERK1/2), and the activities of Pin1 and α-smooth muscle actin (α-SMA) and other oxidative stress parameters were estimated in the heart. The administration of ISO resulted in an increase in cardiac parameters and elevated the heart-to-body weight ratio. Histopathological examination of heart tissues revealed interstitial inflammatory cellular infiltrate and disorganized collagen fiber deposition. In addition, lipid peroxidation products and oxidative stress marker activity in plasma and tissues were significantly increased in the ISO-treated rats. Western blot analysis showed significantly elevated protein levels of phosphorylated Pin1, MEK1/2, ERK1/2 and α-SMA in remodeling hearts. Treatment with juglone following intraperitoneal injection of ISO significantly prevented inflammatory cell infiltration, improved cardiac function, and suppressed oxidative stresses and fibrotic alterations. In conclusion, the results of the present study suggested that the activation of Pin1 promoted cardiac extracellular matrix deposition and oxidative stress damage by regulating the phosphorylation of the MEK1/2-ERK1/2 signaling pathway and the expression of α-SMA. By contrast, the inhibition of Pin1 alleviated cardiac damage and fibrosis in the experimental models, suggesting that Pin1 contributed to the development of cardiac remodeling in ISO-administered rats, and that the inactivation of Pin1 may be a novel therapeutic candidate for the treatment of cardiovascular disease and heart failure.

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 347: The role of Pin1 in chemosensitivity of BRCA1-deficient breast cancers

Abstract BRCA1-deficient breast cancers are largely triple negative (ERα, PR and HER2 negative) and express markers representing the basal-like subgroup. Triple receptor negativity means that there are no targeted therapies available for this subtype, which consequently has the poorest overall survival rates. Given that BRCA1 dysfunction or ‘BRCAness’ is often observed in triple negative breast cancers (TNBCs), it is likely that loss of BRCA1 also plays a key role in their pathogenesis. Therefore to improve TNBC treatment responses, investigation into the underlying biology is required. It has been reported that levels of the prolyl isomerase Pin1 are upregulated in the absence of functional BRCA1, and that levels correlate with tumor grade. Pin1 alters the conformation of target proteins to affect their regulation and function. Many of these target proteins are implicated in key oncogenic signalling pathways, including BRCA1-associated pathways, such as cell cycle and growth regulation, DNA damage response, transcriptional regulation and NFκB signalling. We have observed that Pin1 levels are indeed upregulated in BRCA1-deficent cell lines compared to matched BRCA1 reconstituted cells, and that Pin1 plays a role in their growth rate and responsiveness to chemotherapy. BRCA1-deficient cells are sensitive to DNA damaging agents due to loss of DNA repair functions, notably homologous recombination (HR) repair of double strand breaks. We have identified a correlation between Pin1 levels and sensitivity to PARP inhibitors, which are toxic to HR deficient cells due to synthetic lethality. Ongoing work will establish whether Pin1 levels may be utilized as a biomarker of BRCAness and PARP inhibitor sensitivity. In contrast, during treatment of breast cancer cells with microtubule damaging agents, the presence of BRCA1 is required to promote apoptosis through MEKK3 and JNK, therefore BRCA1 deficient cells are less sensitive. Knockdown of Pin1 in BRCA1-deficient cells sensitizes them to the taxane Paclitaxel, due to repression of the anti-apoptotic protein Mcl-1, while Pin1 knockdown in BRCA1 proficient cells has no effect on sensitivity as Mcl-1 levels are repressed through the MEKK3 pathway. We have also identified that several Src family kinases (SFKs) are upregulated by Pin1 in a BRCA1-deficient background, and may play a role in taxane sensitivity. Pre-treatment with the Src/SFK inhibitor Dasatinib increases cytotoxicity of Paclitaxel, suggesting that combination treatment of Dasatinib and Paclitaxel may be an effective treatment option for BRCA1-deficient breast cancers. In conclusion, we have found that Pin1 may be an important oncogenic mediator downstream of dysfunctional BRCA1 facilitating the viability of TNBCs. We propose that Pin1 could serve as a predictive marker of PARP and Src inhibitor treatments, allowing clinicians to make better informed treatment decisions for this heterogeneous and aggressive subtype of breast cancer. Citation Format: Catherine Knowlson, Paul Mullan, Niamh Buckley. The role of Pin1 in chemosensitivity of BRCA1-deficient breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 347. doi:10.1158/1538-7445.AM2017-347