ABSTRACT: 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. We used microarrays to detail the global gene expression of mammary epithelial cells (MECs) and neuron cells (NCs) in Pin1 KO and WT mice Overall design: Lin-MECs and NCs from two pairs of three month old Pin1 KO and WT littermates were analyzed. Microarray expression profiles were collected using Affymetrix GeneChip Mouse Expression Array 430A.
Project description:Pin1 is a prolyl isomerase that plays an important role in cancer, Alzheimer's disease, and asthma. To understand how Pin1 levels affect mRNA abundance on a genome-wide scale, we knocked down Pin1 by siRNA and used microarray-based gene expression profiling to identify candidate genes that are significantly up- or down-regulated by Pin1 siRNA. The data provide important information on Pin1-responsive genes. We find that genes that are inherently unstable and have short half-lives are targeted by Pin1. Total RNA was obtained from HeLa cells treated with either control siRNA or an siRNA towards Pin1. There were three replicates each for the control and the siRNA-treated cells. The average signal for the three replicates is reported.
Project description:The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma and substitutions in codon 58 stabilize the Myc protein or augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 and Thr 58 provide a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, the role of Pin1 in Myc-induced lymphomagenesis remains unknown. We show here that genetic ablation of Pin1 reduces lymphomagenesis in Eµ-myc transgenic mice. In both Pin1-deficient B-cells and MEFs, the proliferative response to Myc was selectively impaired, with no alterations in Myc-induced apoptosis or mitogen-induced cell cycle entry. This proliferative defect wasn't attributable to alterations in either Ser 62 phosphorylation or Myc-regulated transcription, but to the indirect activation of an Arf-p53 dependent cytostatic response. Pin1 silencing in lymphomas retarded disease progression in mice, making Pin1 an attractive therapeutic target in Myc-driven tumors. RNAseq samples of Pin1+/+ control (n=4), Pin1 -/- control (n=2), Pin1+/+ Eµ-myc pre-tumoral (n=3), and Pin1-/- Eµ-myc pre-tumoral (n=4) B cells.
Project description:Early-stage Alzheimer's disease is characterized by the loss of dendritic spines in the neocortex of the brain. This phenomenon precedes tau pathology, plaque formation, and neurodegeneration and likely contributes to synaptic loss, memory impairment, and behavioral changes in patients. Studies suggest that spine loss is induced by soluble, multimeric Ab42, whose post-synaptic signaling activates the protein phosphatase calcineurin. We investigated how calcineurin causes spine pathology and found that the cis-trans prolyl isomerase Pin1 is a critical downstream target of Ab42/calcineurin signaling. In spines, Pin1 interacts with and is dephosphorylated by calcineurin, which rapidly suppresses its isomerase activity. Pin1 knockout or Ab42 exposure induced mature spine loss in Ab42-treated wild-type cells but had no effect on Pin1 null neurons. The data implicate Pin1 in spine maintenance and synaptic loss in early Alzheimer's disease.
Project description:ATRA was identified as a Pin1 inhibitor via fluorescence polarization-based high throughput screening. We performed microarray expression profiling to demonstrate the similarity between ATRA and Pin1 KD at the genome-wide level APL NB4 cells in response to ATRRA or inducible Pin1 knockdown for 3 days were collected for RNA extraction and hybridization on Affymetrix microarrays. We sought to validate in genome-wide level whether similarity occurred between ATRA and Pin1 knockdown-treated NB4 cells.
Project description:Transforming growth factor (TGF)-β plays crucial roles in embryonic development and adult tissue homeostasis by eliciting various cellular responses in target cells. TGF-β signaling is principally mediated through receptor-activated Smad proteins, which regulate expression of target genes in cooperation with other DNA-binding transcriptionfactors (Smad cofactors). In this study, we found that the basic helix-loop-helix transcription factor Olig1 is a Smad cofactor involved in TGF-b-induced cell motility. Knockdown of Olig1 attenuated TGF-β-induced cell motility in chamber migration and wound healing assays. In contrast, Olig1 knockdown had no effect on bone morphogenetic protein-induced cell motility, TGF-β-induced cytostasis or epithelial-mesenchymal transition. Furthermore, we observed that cooperation of Smad2/3 with Olig1 is regulated by a peptidyl-prolyl cis/trans isomerase, Pin1. TGF-b-induced cell motility, induction of Olig1-regulated genes, and physical interaction between Smad2/3 and Olig1 were all inhibited after knockdown of Pin1, indicating a novel mode of regulation of Smad signaling. We also found that Olig1 interacts with the L3 loop of Smad3. Using a synthetic peptide corresponding to the L3 loop of Smad3, we succeeded in selectively inhibiting TGF-b-induced cell motility. These findings may lead to a new strategy for selective regulation of TGF-b-induced cellular responses. NMuMG cells were transfected with siRNAs (siControl, siOlig1 or siPin1) and treated with or without TGF-b for 1h. We compared genes affected by knockdown of Olig1 and that of Pin1.
Project description:Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but the mechanisms underlying their action and synergy remain elusive. ATRA inhibits APL, breast and liver cancers by targeting isomerase Pin1, a master regulator of oncogenic signaling. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1’s active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, similar to Pin1 CRISPR knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic Pin1 inhibition by ATO and ATRA offers an attractive approach to combating breast and other cancers.
Project description:To discover the abnormal regulatory role of Pin1 in the ovarian cancer, The Pin1 full cDNA was knockIn (KI) the normal ovarian epithelium (Hose), and parallelly Pin1 was knockdown in the Ovarian cancer cells MCAS and sk-ov-3, and then compared the expression profiles of them to discovery the key features regulated by Pin1. Overall design: There are three groups cells used for this expression profile comparison : Hose Pin1 KnockIn (KI) and Hose scramble control; Skov3 Pin1 knockdown (KD) and Scramble control; Mcas Pin1 knockdown and scramble control. The RNA was extracted from well cultured cells using TRIzol reagent, and whole transcriptome gene expression profiling were performed using an Affymetrix Human Gene 1.1 ST whole transcriptome microarray platform.
Project description:To investigate the specific gene expression program by which mutant-p53 and Pin1 control invasion and metastasis in breast cancer cells, we compared the transcriptomic profile of control, mutant-p53 depleted or Pin1 depleted MDA-MB-231 cells. Overall design: MDA-MB-231 cells were transfected twice with siRNA against Pin1, p53 or LacZ as a control. Transfections were performed by using Lifofectamine 2000 (Invitrogen) according to manufacture's procedure. Forty-eight hours after second transfection, samples were then processed for total RNA extraction and hybridization on Affymetrix microarrays. Three biological replicas (A, B, C) were used for each of the three conditions, for a total of 9 samples
Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is phosphorylated at Thr344 and phosphorylation on the C-terminal tail of CK2α is required for interaction with Pin1 protein. The substrate selectivity for protein kinase CK2α was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail or phospho-Thr (pThr) at T344. These semisynthetic proteins were used to determine if the phosphorylation-dependent interaction of CK2α with Pin1 can modulate the substrate selectivity for CK2. The different semisynthetic CK2α proteins (unmodified and pThr344) were tested alone and in the presence of the recombinant Pin1 protein. Pin1 has been shown to interaction with CK2α only when CK2α is phoshorylated on its C-terminal site (including Thr344). In the study presented here, kinase assays were performed using two different semisynthetic CK2α proteins: unmodified C-terminal tail and phospho-Thr (pThr) at 344. The semisynthetic proteins were each tested alone and in the presence of the recombinant Pin1 protein. There were four different kinase conditions and each condition was performed in duplicate.
Project description:From a mechanism-based screening we have identified a novel Pin1 suicide inhibitor, KPT-6566, able to selectively inhibit Pin1 and target it for degradation. We have performed a transcriptiomic analysis comparing Pin1 specific RNA interference in MDA-MB 231 cells with KPT-6566 treatment to assess specificity of the KPT-6566 towards Pin1. Overall design: One cell line was transfected with control siRNA or Pin1 siRNA or treated with KPT-6566 or DMSO. The study comprises 10 experimental points, including duplicate experiments for each siRNA condition and triplicate experiments for the treatment with KPT-6566 or DMSO.