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.

Stereospecific NANOG PEST Stabilization by Pin1.

NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

FASN negatively regulates p65 expression by reducing its stability via Thr254 phosphorylation and isomerization by Pin1.

FASN, the sole cytosolic enzyme responsible for de novo palmitate synthesis in mammalian cells, has been associated with poor prognosis in cancer and shown to cause drug and radiation resistance by upregulating DNA damage repair via suppression of p65 expression. Targeting FASN by repurposing proton pump inhibitors has generated impressive outcomes in triple-negative breast cancer patients. While p65 regulation of DNA damage repair was thought to be due to its suppression of poly(ADP-ribose) polymerase 1 gene transcription, the mechanism of FASN regulation of p65 expression was unknown. In this study, we show that FASN regulates p65 stability by controlling its phosphorylation at Thr254, which recruits the peptidyl-prolyl cis/trans isomerase Pin1 that is known to stabilize many proteins in the nucleus. This regulation is mediated by palmitate, the FASN catalytic product, not by FASN protein per se. This finding of FASN regulation of p65 stability via phosphorylation of Thr254 and isomerization by Pin1 implicates that FASN and its catalytic product palmitate may play an important role in regulating protein stability in general and p65 more specifically.

Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells.

The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

Nuclear Phosphoproteome Reveals Prolyl Isomerase PIN1 as a Modulator of Oncogene-Induced Senescence.

Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex and remain to be fully characterized. In this study, we analyzed the changes in the nuclear proteome and phosphoproteome of human lung fibroblast IMR90 cells during the progression of OIS induced by oncogenic RASG12V activation. We found that most of the differentially regulated phosphosites during OIS contained prolyl isomerase PIN1 target motifs, suggesting PIN1 is a key regulator of several promyelocytic leukemia nuclear body proteins, specifically regulating several proteins upon oncogenic Ras activation. We showed that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence, including p21, p16, and p53 with concomitant accumulation of the protein PML and the dysregulation of promyelocytic leukemia nuclear body formation. Collectively, our data demonstrate that PIN1 plays an important role as a tumor suppressor in response to oncogenic ER:RasG12V activation.

PIN1P1 is activated by CREB1 and promotes gastric cancer progression via interacting with YBX1 and upregulating PIN1.

Long noncoding RNAs (lncRNAs) play critical roles in the carcinogenesis and progression of cancers. However, the role and mechanism of the pseudogene lncRNA PIN1P1 in gastric carcinoma remain unclear. The expression and effects of lncRNA PIN1P1 in gastric cancer were investigated. The transcriptional regulation of CREB1 on PIN1P1 was determined by ChIP and luciferase assays. The mechanistic model of PIN1P1 in gastric cancer was further explored by RNA pull-down, RIP and western blot analysis. PIN1P1 was overexpressed in gastric cancer tissues, and upregulated PIN1P1 predicted poor prognosis in patients. CREB1 was directly combined with the promoter region of PIN1P1 to promote the transcription of PIN1P1. CREB1-mediated enhanced proliferation, migration and invasion could be partially reversed by downregulation of PIN1P1. Overexpressed PIN1P1 promoted the proliferation, migration and invasion of gastric cancer cells, whereas decreased PIN1P1 showed the opposite effects. PIN1P1 directly interacted with YBX1 and promoted YBX1 protein expression, leading to upregulation of PIN1, in which E2F1 may be involved. Silencing of YBX1 during PIN1P1 overexpression could partially rescue PIN1 upregulation. PIN1, the parental gene of PIN1P1, was elevated in gastric cancer tissues, and its upregulation was correlated with poor patient outcomes. PIN1 facilitated gastric cancer cell proliferation, migration and invasion. To sum up, CREB1-activated PIN1P1 could promote gastric cancer progression through YBX1 and upregulating PIN1, suggesting that it is a potential target for gastric cancer.

The PIN1-YTHDF1 axis promotes breast tumorigenesis via the m6A-dependent stabilization of AURKA mRNA.

The post-transcriptional processing of N6-methyladenosine (m6A)-modified mRNA by YTH domain-containing family protein 1 (YTHDF1) plays a crucial role in the regulation of gene expression. Although YTHDF1 expression is frequently upregulated in breast cancer, the regulatory mechanisms for this remain unclear. In this study, we examined the role of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) in regulating YTHDF1 stability in breast cancer cells. The WW domain of PIN1 interacted with YTHDF1 in a phosphorylation-dependent manner. Additionally, PIN1 overexpression increased YTHDF1 stability by preventing ubiquitin-dependent proteasomal degradation. Furthermore, using the MS2-tagged RNA pull-down assay, we identified Aurora kinase A (AURKA) mRNA as a bona fide substrate of YTHDF1. PIN1-mediated YTHDF1 stabilization increased the stability of AURKA mRNA in an m6A-dependent manner. Furthermore, YTHDF1 knockout reduced AURKA protein expression levels, resulting in anticancer effects in breast cancer cells, including decreased cell proliferation, cell cycle arrest at the G0/G1 phase, apoptotic cell death, and decreased spheroid formation. The anticancer effects induced by YTHDF1 knockout were reversed by AURKA overexpression. Similarly, the knockout of PIN1 produced comparable anticancer effects to those observed in YTHDF1-knockout cells, and these effects were reversed upon overexpression of YTHDF1. In conclusion, the findings of our study suggest that increased YTHDF1 stability induced by PIN1 promotes breast tumorigenesis via the stabilization of AURKA mRNA. Targeting the PIN1/YTHDF1 axis may represent a novel therapeutic strategy for breast cancer.

Oxygen-independent stabilization of HIF-2α in breast cancer through direct interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

The expression of prolyl isomerase Pin1 is expanded in the skin of patients with atopic dermatitis and facilitates IL-33 expression in HaCaT cells.

Atopic dermatitis (AD) is attributable to both a genetic predisposition and environmental factors. Among numerous cytokines involved in the pathogenesis of AD, interleukin-33 (IL-33), reportedly escaping exocytotically in response to a scratch, is abundantly expressed in the skin tissues of patients with AD and is postulated to induce inflammatory and autoimmune responses. In this study, we first demonstrated that peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1), a unique enzyme which isomerizes the proline residues of target proteins, is abundantly expressed in keratinocytes, and that the areas where it is present in the skin tissues of AD patients became expanded due to hyperkeratosis. Thus, we investigated the effects of Pin1 on the regulation of IL-33 expression using the human keratinocyte cell line HaCaT. Interestingly, silencing of the Pin1 gene or treatment with Pin1 inhibitors dramatically reduced IL-33 expressions in HaCaT cells, although Pin1 overexpression did not elevate it. Subsequently, we showed that Pin1 binds to STAT1 and the nuclear factor-kappaB (NF-κB) subunit p65. Silencing the Pin1 gene with small interfering RNAs significantly reduced the phosphorylation of p65, while no marked effects of Pin1 on the STAT1 pathway were detected. Thus, it is likely that Pin1 contributes to increased expression of IL-33 via the NF-κB subunit p65 in HaCaT cells, at least modestly. Nevertheless, further study is necessary to demonstrate the pathogenic roles of Pin1 and IL-33 in AD development.

MicroRNA-150-5p inhibits the proliferation and invasion of human larynx epidermiod cancer cells though regulating peptidyl-prolyl cis/trans isomerase.

OBJECTIVE: This study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1). METHODS: Firstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase reporter assay, followed by their correlation analysis. Besides, after interfering with miR-150-5p or PIN1 expression in HEp-2 cells, CCK-8, cell colony formation assay, and transwell assay were utilized to detect the proliferation, viability, and invasion of cells, respectively. Subsequently, the protein levels of MMP-2, MMP-9, and EMT-related proteins in HEp-2 cells were checked by Western blot. RESULTS: Expression of miR-150-5p was down-regulated in LSCC tissues and HEp-2 cells. Moreover, miR-150-5p suppressed proliferation and invasion of HEp-2 cells, affected protein expression related to MMP and EMT, thereby inhibiting development of cancer. The expression of PIN1 was significantly increased in cancer tissues and HEp-2 cells, and there was a targeted relationship and negative correlation between miR-150-5p and PIN1 in cancer tissue. However, overexpression of PIN1 could reverse the effect of miR-150-5p on the proliferation and invasion of HEp-2 cells. CONCLUSION: In a nutshell, there is a targeted relationship between PIN1 and miR-150-5p. Besides, miR-150-5p can inhibit the proliferation and invasion of HEp-2 cells by regulating the expression of PIN1.

The metabolic crosstalk between PIN1 and the tumour microenvironment.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is a member of a family of peptidyl-prolyl isomerases that specifically recognizes and binds phosphoproteins, catalyzing the rapid cis-trans isomerization of phosphorylated serine/threonine-proline motifs, which leads to changes in the structures and activities of the targeted proteins. Through this complex mechanism, PIN1 regulates many hallmarks of cancer including cell autonomous metabolism and the crosstalk with the cellular microenvironment. Many studies showed that PIN1 is largely overexpressed in cancer turning on a set of oncogenes and abrogating the function of tumor suppressor genes. Among these targets, recent evidence demonstrated that PIN1 is involved in lipid and glucose metabolism and accordingly, in the Warburg effect, a characteristic of tumor cells. As an orchestra master, PIN1 finely tunes the signaling pathways allowing cancer cells to adapt and take advantage from a poorly organized tumor microenvironment. In this review, we highlight the trilogy among PIN1, the tumor microenvironment and the metabolic program rewiring.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Key role of PIN1 in telomere maintenance and oncogenic behavior in a human glioblastoma model.

PIN1 is the only known enzyme capable of recognizing and isomerizing the phosphorylated Serine/Threonine­Proline motif. Through this mechanism, PIN1 controls diverse cellular functions, including telomere maintenance. Both PIN1 overexpression and its involvement in oncogenic pathways are involved in several cancer types, including glioblastoma (GBM), a lethal disease with poor therapeutic resources. However, knowledge of the role of PIN1 in GBM is limited. Thus, the present work aimed to study the role of PIN1 as a telomere/telomerase regulator and its contribution to tumor biology. PIN1 knockout (KO) LN­229 cell variant using CRISPR/Cas9 was developed and compared with PIN1 LN­229 expressing cells. To study the effect of PIN1 absence, status of NF­κB pathway was evaluated by luciferase reporter gene assay and quantitative PCR. Results revealed that PIN1 deletion in GBM cells diminished the active levels of NF­κB and decrease the transcription of il­8 and htert genes. Then, telomere/telomerase related processes were studied by RQ­TRAP assay and telomere length determination by qPCR, obtaining a reduction both in telomerase activity as in telomere length in PIN1 KO cells. In addition, measurement of SA ß­galactosidase and caspase­3 activities revealed that loss of PIN1 triggers senescence and apoptosis. Finally, migration, cell cycle progression and tumorigenicity were studied by flow cytometry/western blot, Transwell assay and in vivo experiments, respectively. PIN1 deletion decreased migration as well as cell cycle progression by increasing doubling time and also resulted in the loss of LN­229 cell ability to form tumors in mice. These results highlight the role of PIN1 in telomere homeostasis and GBM progression, which supports PIN1 as a potential molecular target for the development of novel therapeutic agents for GBM treatment.

Prolyl isomerase Pin1 promotes extracellular matrix production in hepatic stellate cells through regulating formation of the Smad3-TAZ complex.

Hepatic stellate cells (HSCs) produce extracellular matrixes (ECMs), such as collagen and fibronectin, in response to stimulation with transforming growth factor ß (TGFß). The massive ECM accumulation in the liver due to HSCs causes fibrosis which eventually leads to hepatic cirrhosis and hepatoma development. However, details of the mechanisms underlying continuous HSC activation are as yet poorly understood. We thus attempted to elucidate the role of Pin1, one of the prolyl isomerases, in the underlying mechanism(s), using the human HSC line LX-2. Treatment with Pin1 siRNAs markedly alleviated the TGFß-induced expressions of ECM components such as collagen 1a1/2, smooth muscle actin and fibronectin at both the mRNA and the protein level. Pin1 inhibitors also decreased the expressions of fibrotic markers. In addition, it was revealed that Pin1 associates with Smad2/3/4, and that four Ser/Thr-Pro motifs in the linker domain of Smad3 are essential for binding with Pin1. Pin1 significantly regulated Smad-binding element transcriptional activity without affecting Smad3 phosphorylations or translocation. Importantly, both Yes-associated protein (YAP) and WW domain-containing transcription regulator (TAZ) also participate in ECM induction, and upregulate Smad3 activity rather than TEA domain transcriptional factor transcriptional activity. Although Smad3 interacts with both TAZ and YAP, Pin1 facilitates the Smad3 association with TAZ, but not that with YAP. In conclusion, Pin1 plays pivotal roles in ECM component productions in HSCs through regulation of the interaction between TAZ and Smad3, and Pin1 inhibitors may have the potential to ameliorate fibrotic diseases.

METTL3 stabilization by PIN1 promotes breast tumorigenesis via enhanced m6A-dependent translation.

Methyltransferase-like 3 (METTL3) is the catalytic subunit of the N6-adenosine methyltransferase complex responsible for N6-methyladenosine (m6A) modification of mRNA in mammalian cells. Although METTL3 expression is increased in several cancers, the regulatory mechanisms are unclear. We explored the regulatory roles of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) in METTL3 stability and m6A modification of mRNA. PIN1 interacted with METTL3 and prevented its ubiquitin-dependent proteasomal and lysosomal degradation. It stabilized METTL3, which increased the m6A modification of transcriptional coactivator with PDZ-binding motif (TAZ) and epidermal growth factor receptor (EGFR) mRNA, resulting in their efficient translation. PIN1 knockout altered the distribution of TAZ and EGFR mRNA from polysomes into monosomes. Inhibition of MEK1/2 kinases and PIN1 destabilized METTL3, which impeded breast cancer cell proliferation and induced cell cycle arrest at the G0/G1 phases. METTL3 knockout reduced PIN1 overexpression-induced colony formation in MCF7 cells and enhanced tumor growth in 4T1 cells in an orthotopic mouse model. In clinical settings, METTL3 expression significantly increased with tumor progression and was positively correlated with PIN1 expression in breast cancer tissues. Thus, PIN1 plays a regulatory role in mRNA translation, and the PIN1/METTL3 axis may be an alternative therapeutic target in breast cancer.

PIN1 and CDK1 cooperatively govern pVHL stability and suppressive functions.

The VHL protein (pVHL) functions as a tumor suppressor by regulating the degradation or activation of protein substrates such as HIF1α and Akt. In human cancers harboring wild-type VHL, the aberrant downregulation of pVHL is frequently detected and critically contributes to tumor progression. However, the underlying mechanism by which the stability of pVHL is deregulated in these cancers remains elusive. Here, we identify cyclin-dependent kinase 1 (CDK1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) as two previously uncharacterized regulators of pVHL in multiple types of human cancers harboring wild-type VHL including triple-negative breast cancer (TNBC). PIN1 and CDK1 cooperatively modulate the protein turnover of pVHL, thereby conferring tumor growth, chemotherapeutic resistance and metastasis both in vitro and in vivo. Mechanistically, CDK1 directly phosphorylates pVHL at Ser80, which primes the recognition of pVHL by PIN1. PIN1 then binds to phosphorylated pVHL and facilitates the recruitment of the E3 ligase WSB1, therefore targeting pVHL for ubiquitination and degradation. Furthermore, the genetic ablation or pharmacological inhibition of CDK1 by RO-3306 and PIN1 by all-trans retinoic acid (ATRA), the standard care for Acute Promyelocytic Leukemia could markedly suppress tumor growth, metastasis and sensitize cancer cells to chemotherapeutic drugs in a pVHL dependent manner. The histological analyses show that PIN1 and CDK1 are highly expressed in TNBC samples, which negatively correlate with the expression of pVHL. Taken together, our findings reveal the previous unrecognized tumor-promoting function of CDK1/PIN1 axis through destabilizing pVHL and provide the preclinical evidence that targeting CDK1/PIN1 is an appealing strategy in the treatment of multiple cancers with wild-type VHL.

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation.

BACKGROUND: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood. METHODS: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems. RESULTS: We observed positive associations between L1 and activated TGFß-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFß-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFß-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p. DISCUSSION: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.