Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease.

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.

Function and regulation of cis P-tau in the pathogenesis and treatment of conventional and nonconventional tauopathies.

Conventional tauopathies are a group of disease characterized by tau inclusions in the brains, including Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and certain types of frontotemporal dementia (FTD), among which AD is the most prevalent. Extensive post-translational modifications, especially hyperphosphorylation, and abnormal aggregation of tau protein underlie tauopathy. Cis-trans isomerization of protein plays an important role in protein folding, function, and degradation, which is regulated by peptidyl-proline isomerases (PPIases). Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), the only PPIase found to isomerize Pro following phosphorylated Ser or Thr residues, alters phosphorylated tau protein conformation at pT231-P motif. The cis P-tau but not trans P-tau serves as an early driver of multiple neurodegenerative disease, encompassing AD, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and vascular contributions to cognitive impairment and dementia (VCID). Cis but not trans P-tau is resistant to protein dephosphorylation and degradation, and also prone to protein aggregation. Cis P-tau loses its ability to stabilize microtubule, causing and spreading tauopathy mainly in axons, a pathological process called cistauosis. The conformation-specific monoclonal antibody that targets only the cis P-tau serves as a very early diagnosis method and a potential treatment of not only conventional tauopathies but also nonconventional tauopathies such as VCID, with clinical trials ongoing. Notably, cis P-tau antibody is the only clinical-stage Alzheimer's therapeutic that has shown the efficacy in animal models of not only AD but also TBI and stroke, which are very early stages of dementia. Here we review the identification and pathological consequences of cis pt231-tau, the role of its regulator Pin1, as well as the clinical implication of cis pt231-tau conformation-specific antibody in conventional and nonconventional tauopathies.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

ATR Plays a Direct Antiapoptotic Role at Mitochondria, which Is Regulated by Prolyl Isomerase Pin1.

ATR, a PI3K-like protein kinase, plays a key role in regulating DNA damage responses. Its nuclear checkpoint kinase function is well documented, but little is known about its function outside the nucleus. Here we report that ATR has an antiapoptotic activity at mitochondria in response to UV damage, and this activity is independent of its hallmark checkpoint/kinase activity and partner ATRIP. ATR contains a BH3-like domain that allows ATR-tBid interaction at mitochondria, suppressing cytochrome c release and apoptosis. This mitochondrial activity of ATR is downregulated by Pin1 that isomerizes ATR from cis-isomer to trans-isomer at the phosphorylated Ser428-Pro429 motif. However, UV inactivates Pin1 via DAPK1, stabilizing the pro-survival cis-isomeric ATR. In contrast, nuclear ATR remains in the trans-isoform disregarding UV. This cytoplasmic response of ATR may provide a mechanism for the observed antiapoptotic role of ATR in suppressing carcinogenesis and its inhibition in sensitizing anticancer agents for killing of cancer cells.

Identification of a potent and selective covalent Pin1 inhibitor.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

The Pin1-CaMKII-AMPA Receptor Axis Regulates Epileptic Susceptibility.

Pin1 is a unique isomerase that regulates protein conformation and function after phosphorylation. Pin1 aberration contributes to some neurological diseases, notably Alzheimer's disease, but its role in epilepsy is not fully understood. We found that Pin1-deficient mice had significantly increased seizure susceptibility in multiple chemical inducing models and developed age-dependent spontaneous epilepsy. Electrophysiologically, Pin1 ablation enhanced excitatory synaptic transmission to prefrontal cortex (PFC) pyramidal neurons without affecting their intrinsic excitability. Biochemically, Pin1 ablation upregulated AMPA receptors and GluA1 phosphorylation by acting on phosphorylated CaMKII. Clinically, Pin1 was decreased significantly, whereas phosphorylated CaMKII and GluA1 were increased in the neocortex of patients with epilepsy. Moreover, Pin1 expression restoration in the PFC of Pin1-deficient mice using viral gene transfer significantly reduced phosphorylated CaMKII and GluA1 and effectively suppressed their seizure susceptibility. Thus, Pin1-CaMKII-AMPA receptors are a novel axis controlling epileptic susceptibility, highlighting attractive new therapeutic strategies.

Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy.

Traumatic brain injury (TBI), characterized by acute neurological dysfunction, is one of the best known environmental risk factors for chronic traumatic encephalopathy and Alzheimer's disease, the defining pathologic features of which include tauopathy made of phosphorylated tau protein (P-tau). However, tauopathy has not been detected in the early stages after TBI, and how TBI leads to tauopathy is unknown. Here we find robust cis P-tau pathology after TBI in humans and mice. After TBI in mice and stress in vitro, neurons acutely produce cis P-tau, which disrupts axonal microtubule networks and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, which we term 'cistauosis', appears long before other tauopathy. Treating TBI mice with cis antibody blocks cistauosis, prevents tauopathy development and spread, and restores many TBI-related structural and functional sequelae. Thus, cis P-tau is a major early driver of disease after TBI and leads to tauopathy in chronic traumatic encephalopathy and Alzheimer's disease. The cis antibody may be further developed to detect and treat TBI, and prevent progressive neurodegeneration after injury.

Melatonin directly binds and inhibits death-associated protein kinase 1 function in Alzheimer's disease.

Death-associated protein kinase 1 (DAPK1) is upregulated in the brains of human Alzheimer's disease (AD) patients compared with normal subjects, and aberrant DAPK1 regulation is implicated in the development of AD. However, little is known about whether and how DAPK1 function is regulated in AD. Here, we identified melatonin as a critical regulator of DAPK1 levels and function. Melatonin significantly decreases DAPK1 expression in a post-transcriptional manner in neuronal cell lines and mouse primary cortical neurons. Moreover, melatonin directly binds to DAPK1 and promotes its ubiquitination, resulting in increased DAPK1 protein degradation through a proteasome-dependent pathway. Furthermore, in tau-overexpressing mouse brain slices, melatonin treatment and the inhibition of DAPK1 kinase activity synergistically decrease tau phosphorylation at multiple sites related to AD. In addition, melatonin and DAPK1 inhibitor dramatically accelerate neurite outgrowth and increase the assembly of microtubules. Mechanistically, melatonin-mediated DAPK1 degradation increases the activity of Pin1, a prolyl isomerase known to play a protective role against tau hyperphosphorylation and tau-related pathologies. Finally, elevated DAPK1 expression shows a strong correlation with the decrease in melatonin levels in human AD brains. Combined, these results suggest that DAPK1 regulation by melatonin is a novel mechanism that controls tau phosphorylation and function and offers new therapeutic options for treating human AD.

Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase.

Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.

Targeting PIN1 exerts potent antitumor activity in pancreatic ductal carcinoma via inhibiting tumor metastasis.

The human prolyl isomerase PIN1, best known for its association with carcinogenesis, has recently been indicated in the disease of pancreatic ductal adenocarcinoma (PDAC). However, the functions of PIN1 and the feasibility of targeting PIN1 in PDAC remain elusive. For this purpose, we examined the expression of PIN1 in cancer, related paracarcinoma and metastatic cancer tissues by immunohistochemistry and analyzed the associations with the pathogenesis of PDAC in 173 patients. The functional roles of PIN1 in PDAC were explored in vitro and in vivo using both genetic and chemical PIN1 inhibition. We showed that PIN1 was upregulated in pancreatic cancer and metastatic tissues. High PIN1 expression is significantly association with poor clinicopathological features and shorter overall survival and disease-free survival. Further stratified analysis showed that PIN1 phenotypes refined prognostication in PDAC. Inhibition of PIN1 expression with RNA interference or with all trans retinoic acid decreased not only the growth but also the migration and invasion of PDAC cells through regulating the key molecules of multiple cancer-driving pathways, simultaneously resulting in cell cycle arrest and mesenchymal-epithelial transition in vitro. Furthermore, genetic and chemical PIN1 ablation showed dramatic inhibition of the tumorigenesis and metastatic spread and then reduced the tumor burden in vivo. We provided further evidence for the use of PIN1 as a promising therapeutic target in PDAC. Genetic and chemical PIN1 ablation exerted potent antitumor effects through blocking multiple cancer-driving pathways in PDAC. More potent and specific PIN1 targeted inhibitors could be exploited to treat this aggressive cancer.

Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/ß-catenin oncogenic pathways.

Gastric cancer is the second leading cause of cancer-related mortality and the fourth most common cancer globally. High intratumor heterogeneity of advanced gastric cancer poses great challenges to targeted therapy due to simultaneous activation of many redundant cancer-driving pathways. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 inhibition exerts anticancer activity by blocking multiple cancer-driving pathways in some cancers, but its role in gastric cancer is not fully understood. Here we detected Pin1 protein expression in 1065 gastric cancer patients and paired normal tissues using immunohistochemistry and Western blot, and then examined the effects of Pin1 overexpression, and genetic and chemical Pin1 inhibition using Pin1 short hairpin RNA or small molecule inhibitor all-trans retinoic acid (ATRA) on tumorigenesis of human gastric cancer in vitro and in vivo, followed by biochemical analyses to elucidate Pin1 regulated oncogenic pathways. We found that Pin1 was significantly overexpressed in primary and metastasized tumors, with Pin1 overexpression being correlated with advanced stage and poor prognosis. Furthermore, whereas Pin1 overexpression promoted the transformed phenotype in immortalized and nontransformed human gastric cells, either genetic or chemical Pin1 inhibition in multiple human gastric cancer cells potently suppressed cell growth, G1/S transition and colony formation in vitro, as well as tumor growth in xenograft tumor models in vivo, which were further supported by downregulation of multiple key oncoproteins in PI3K/AKT and Wnt/ß-catenin signaling pathways. These results not only provide the first evidence for a critical role of Pin1 in the tumorigenesis of gastric cancer but also suggest that targeting Pin1 using ATRA or other inhibitors offers an effective new therapeutic approach for treating advanced gastric cancer.

The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease.

Protein phosphorylation regulates many cellular processes by causing changes in protein conformation. The prolyl isomerase PIN1 has been identified as a regulator of phosphorylation signalling that catalyses the conversion of specific phosphorylated motifs between the two completely distinct conformations in a subset of proteins. PIN1 regulates diverse cellular processes, including growth-signal responses, cell-cycle progression, cellular stress responses, neuronal function and immune responses. In line with the diverse physiological roles of PIN1, it has also been linked to several diseases that include cancer, Alzheimer's disease and asthma, and thus it might represent a novel therapeutic target.

Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function.

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.

Death-Associated Protein Kinase 1 Phosphorylation in Neuronal Cell Death and Neurodegenerative Disease.

Regulated neuronal cell death plays an essential role in biological processes in normal physiology, including the development of the nervous system. However, the deregulation of neuronal apoptosis by various factors leads to neurodegenerative diseases such as ischemic stroke and Alzheimer's disease (AD). Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine (Ser/Thr) protein kinase that activates death signaling and regulates apoptotic neuronal cell death. Although DAPK1 is tightly regulated under physiological conditions, DAPK1 deregulation in the brain contributes to the development of neurological disorders. In this review, we describe the molecular mechanisms of DAPK1 regulation in neurons under various stresses. We also discuss the role of DAPK1 signaling in the phosphorylation-dependent and phosphorylation-independent regulation of its downstream targets in neuronal cell death. Moreover, we focus on the major impact of DAPK1 deregulation on the progression of neurodegenerative diseases and the development of drugs targeting DAPK1 for the treatment of diseases. Therefore, this review summarizes the DAPK1 phosphorylation signaling pathways in various neurodegenerative diseases.

FAK phosphorylation by ERK primes ras-induced tyrosine dephosphorylation of FAK mediated by PIN1 and PTP-PEST.

Activated Ras has been found in many types of cancer. However, the mechanism underlying Ras-promoted tumor metastasis remains unclear. We demonstrate here that activated Ras induces tyrosine dephosphorylation and inhibition of FAK mediated by the Ras downstream Fgd1-Cdc42-PAK1-MEK-ERK signaling cascade. ERK phosphorylates FAK S910 and recruits PIN1 and PTP-PEST, which colocalize with FAK at the lamellipodia of migrating cells. PIN1 binding and prolyl isomerization of FAK cause PTP-PEST to interact with and dephosphorylate FAK Y397. Inhibition of FAK mediated by this signal relay promotes Ras-induced cell migration, invasion, and metastasis. These findings uncover the importance of sequential modification of FAK-by serine phosphorylation, isomerization, and tyrosine dephosphorylation--in the regulation of FAK activity and, thereby, in Ras-related tumor metastasis.

Pin1 dysregulation helps to explain the inverse association between cancer and Alzheimer's disease.

BACKGROUND: Pin1 is an intracellular signaling molecule which plays a critical but opposite role in the pathogenesis of Alzheimer's disease (AD) and many human cancers. SCOPE OF REVIEW: We review the structure and function of the Pin1 enzyme, the diverse roles it plays in cycling cells and neurons, the epidemiologic evidence for the inverse association between cancer and AD, and the potential therapeutic implications of Pin1-based therapies. MAJOR CONCLUSIONS: Pin1 is a unique enzyme that has effects on the function of target proteins by "twisting" them into different shapes. Cycling cells use Pin1 to help coordinate cell division. It is over-expressed and/or activated by multiple mechanisms in many common human cancers, and acts on multiple signal pathways to promote tumorigenesis. Inhibition of Pin1 in animal models has profound anti-tumor effects. In contrast, Pin1 is down-regulated or inactivated by multiple mechanisms in AD brains. The absence of Pin1 impairs tau function and amyloid precursor protein processing, leading to tangle- and amyloid-related pathologies and neurodegeneration in an age-dependent manner, resembling human AD. We have developed cis and trans conformation-specific antibodies to provide the first direct evidence that tau exists in distinct cis and trans conformations and that Pin1 accelerates its cis to trans conversion, thereby protecting against tangle formation in AD. GENERAL SIGNIFICANCE: Available studies on Pin1 suggest that cancer and AD may share biological pathways that are deregulated in different directions. Pin1 biology opens exciting preventive and therapeutic horizons for both cancer and neurodegeneration. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins.

Pin1 is a highly conserved enzyme that only isomerizes specific phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Such conformational changes represent a novel and tightly controlled signaling mechanism regulating a spectrum of protein activities in physiology and disease; often through phosphorylation-dependent, ubiquitin-mediated proteasomal degradation. In this review, we summarize recent advances in elucidating the role and regulation of Pin1 in controlling protein stability. We also propose a mechanism by which Pin1 functions as a molecular switch to control the fates of phosphoproteins. We finally stress the need to develop tools to visualize directly Pin1-catalyzed protein conformational changes as a way to determine their roles in the development and treatment of human diseases.

Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer's disease.

The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.