P27 Kip1 mRNA Research Articles

MiRNA-221 and -222 increase in exosomes upon insulin stimulation of human vascular smooth muscle cells lacking the insulin-like growth factor 1 receptor

As a result of increased vascular inflammation, patients with Type 2 diabetes experience a near 4-fold increase in their risk for heart attack and stroke. miR-221 and -222 are two microRNAs that are upregulated in the vasculature of Type 2 diabetic patients. miR-221/222 act by downregulating the cyclin-dependent kinase inhibitor, p27Kip1, in vascular smooth muscle cells (VSMCs) and exosome-mediated proinflammatory effects on endothelial cells and macrophages. Loss of the insulin-like growth factor receptor (IGFR) in VSMCs can unmask the insulin receptor (IR) and increase insulin signaling. We have demonstrated that the loss of IGFR, coupled with physiological insulin stimulation, promotes an increased expression of miR-221/-222 in murine VSMCs. Respectively, miR-221/222 content in VSMC-derived exosomes increases and p27Kip1 mRNA expression decreases in VSMCs. We asked whether the effect of loss of IGFR in human coronary artery smooth muscle cells (HCASMCs) would also lead to an increase in miR-221/-222 content of VSMC-derived exosomes. Transfection with a CRISPR-cas9 plasmid targeting IGFR resulted in a 50% decrease in IGFR mRNA in HCASMCs, when compared to transfection with negative control CRISPR plasmids. Droplet digital PCR was used to measure expression of miR-221 and miR-222 in the exosomes derived from both sets of HCASMCs. There was no change in the miR-221/222 content of exosomes derived from HCASMCs transfected with the negative control CRISPR following stimulation with 1nM insulin (7.27± 4.75 vs. 11.33 ± 1.09 copies of miR-221 and 6.43 ± 0.43 vs. 4.68 ± 0.02 copies of miR-222). In contrast, there was a 2 to 3-fold increase in miR-221/222 content in exosomes that were derived from HCASMCs with IGFR knockdown following stimulation with 1nM insulin (14.96 ± 0.3 vs. 35.66 ± 3.08 copies of miR-221 and 9.12 ± 1.19 vs.15.44 ± 5.36 copies of miR-222). We conclude that loss of IGFR coupled with physiological insulin stimulation increases the miR-221/222 content of exosomes of HCASMCs, recapitulating the effects of IGFR loss seen in murine VSMCs as well as the effects of diabetes on VSMCs. This model system will allow additional studies connecting the loss of IGFR to the cardiovascular complications of diabetes. Tulane University School of Medicine, Biomedical Sciences Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Salidroside Inhibits Reactive Astrogliosis and Glial Scar Formation in Late Cerebral Ischemia via the Akt/GSK-3β Pathway.

Cerebral ischemia leads to reactive astrogliosis and glial scar formation. Glial scarring can impede functional restoration during the recovery phase of stroke. Salidroside has been shown to have neuroprotective effects after ischemic stroke, but its impact on long-term neurological recovery, especially whether it regulates reactive astrogliosis and glial scar formation, is unclear. In this study, male adult C57/BL6 mice were subjected to transient cerebral ischemia injury followed by intravenous salidroside treatment. Primary astrocytes were treated with lipopolysaccharide (LPS) or conditioned medium from cultured primary neurons subjected to oxygen-glucose deprivation (CM-OGD). Salidroside significantly improved long-term functional outcomes following ischemic stroke in the rotarod and corner tests. It also reduced brain glial scar volume and decreased expression of the glial scar marker, glial fibrillary acidic protein (GFAP) and inhibited astrocyte proliferation. In primary astrocyte cultures, salidroside protected astrocytes from CM-OGD injury-induced reactive astroglial proliferation, increasing the percentage of cells in G0/G1 phase and reducing the S populations. The inhibitory effect of salidroside on the cell cycle was related to downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA expression and increased p27Kip1 mRNA expression. Similar results were found in the LPS-stimulated injury model in astroglial cultures. Western blot analysis demonstrated that salidroside attenuated the CM-OGD-induced upregulation of phosphorylated Akt and glycogen synthase kinase 3β (GSK-3β). Taken together, these results suggested that salidroside can inhibit reactive astrocyte proliferation, ameliorate glial scar formation and improve long-term recovery, probably through its effects on the Akt/GSK-3β pathway.

Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3’UTRs in cisplatin treated cells

ABSTRACTPost-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3ʹUTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell’s response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3ʹUTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

KLF5 regulated lncRNA RP1 promotes the growth and metastasis of breast cancer via repressing p27kip1 translation

Increasing evidence suggest that lncRNAs (long noncoding RNAs) play important roles in human cancer. Breast cancer is a heterogeneous disease and the potential involvement of lncRNAs in breast cancer remains unexplored. In this study, we characterized a novel lncRNA, RP1-5O6.5 (termed as RP1). We found that RP1 was highly expressed in breast cancer and predicted poor prognosis of breast cancer patients. Gain-of-function and loss-of-function assays showed that RP1 promoted the proliferation and metastasis of breast cancer cells in vitro and in vivo. Mechanistically, RP1 maintained the EMT and stemness states of breast cancer cells via repressing p27kip1 protein expression. RP1 combined with the complex p-4E-BP1/eIF4E to prevent eIF4E from interacting with eIF4G, therefore attenuating the translational efficiency of p27kip1 mRNA. Furthermore, we found that p27kip1 evidently downregulated Snail1 but not ZEB1 to inhibit invasion of breast cancer cells. Kruppel-like factor 5 (KLF5) was positively correlated with RP1 in breast cancer tissues. Moreover, we demonstrated that KLF5 recruited p300 to the RP1 promoter to enhance RP1 expression. Taken together, our findings demonstrated that KLF5-regulated RP1 plays an oncogenic role in breast cancer by suppressing p27kip1, providing support for the clinical investigation of therapeutic approaches focusing on RP1.

PCBP1 depletion promotes tumorigenesis through attenuation of p27Kip1 mRNA stability and translation

BackgroundPoly C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs. Depletion of PCBP1 is implicated in various carcinomas, but the underlying mechanism in tumorigenesis remains elusive.MethodsWe performed a transcriptome-wide screen to identify novel bounding mRNA of PCBP1. The bind regions between PCBP1 with target mRNA were investigated by using point mutation and luciferase assay. Cell proliferation, cell cycle, tumorigenesis and cell apoptosis were also evaluated in ovary and colon cancer cell lines. The mechanism that PCBP1 affects p27 was analyzed by mRNA stability and ribosome profiling assays. We analyzed PCBP1 and p27 expression in ovary, colon and renal tumor samples and adjacent non-tumor tissues using RT-PCR, Western Blotting and immunohistochemistry. The prognostic significance of PCBP1 and p27 also analyzed using online databases.ResultsWe identified cell cycle inhibitor p27Kip1 (p27) as a novel PCBP1-bound transcript. We then demonstrated that binding of PCBP1 to p27 3’UTR via its KH1 domain mainly stabilizes p27 mRNA, while enhances its translation to fuel p27 expression, prior to p27 protein degradation. The upregulated p27 consequently inhibits cell proliferation, cell cycle progression and tumorigenesis, whereas promotes cell apoptosis under paclitaxel treatment. Conversely, knockdown of PCBP1 in turn compromises p27 mRNA stability, leading to lower p27 level and tumorigenesis in vivo. Moreover, forced depletion of p27 counteracts the tumor suppressive ability of PCBP1 in the same PCBP1 over-expressing cells. Physiologically, we showed that decreases of both p27 mRNA and its protein expressions are well correlated to PCBP1 depletion in ovary, colon and renal tumor samples, independent of the p27 ubiquitin ligase Skp2 level. Correlation of PCBP1 with p27 is also found in the tamoxifen, doxorubincin and lapatinib resistant breast cancer cells of GEO database.ConclusionOur results thereby indicate that loss of PCBP1 expression firstly attenuates p27 expression at post-transcriptional level, and subsequently promotes carcinogenesis. PCBP1 could be used as a diagnostic marker to cancer patients.

Abstract 4305: TET increases p27Kip1 protein stability and p21WAF1/Cip1 promoter activity to block deregulated cell cycle progression in pancreatic cancer cells

Abstract Introduction: Pancreatic cancer (PaCa) is the fourth cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PaCa are ineffective thus there is an urgent need for development of new therapeutic agents for its treatment. Owing to its mutational landscape, which includes mutated KRAS and p53 these cells have a deregulated G1 checkpoint of cell cycle and thus confer high proliferation rate and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression in PaCa cells at G1/S boundary in vitro and in vivo. In the present study we explored potential mechanism of TET induced cell cycle arrest in two PaCa cells with either wild type or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G1/S transition. Method: PaCa cell lines: BxPC3 (PDAC) and MiaPaCa2 (Pancreatic Carcinoma) were used. Cells were treated with various concentrations of TET for different time points. Cell growth was checked using the MTT assay. Cell cycle was analyzed after PI staining via Flow cytometry. Changes in protein levels were assessed by Western Blot analysis. mRNA changes were evaluated by real time PCR. Promoter activity was checked by luciferase reporter assay. Knockdown was achieved by lentiviral transduction. Results: Treatment of PaCa cells with TET resulted in decreased levels of cyclin D1 and phospho-Rb, two critical proteins required for G1/S transition and increased levels of p21WAF1/Cip1 and p27Kip1 proteins. Real time PCR revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27Kip1 mRNA levels. The cycloheximide chase showed that TET treatment increases p27Kip1 protein levels by improving its protein stability (t1/2 6h vs 2h: TET treated vs control). Further studies into the mechanism revealed that treatment of PaCa cells with TET resulted in decreased protein levels of Skp2 E3 ligase which is specific for degrading p27Kip1 protein during the cell cycle. Moreover TET also increased the p21WAF1/Cip1 promoter activity accounting for increased mRNA levels of p21WAF1/Cip1. These data suggest a notable mechanism by which TET regulates expression of critical cell cycle regulators at both transcriptional and post-translational level. Such a mechanism would provide for cell cycle block in PaCa cells following TET treatment. Conclusion: TET halts deregulated cell cycle in PaCa cells in part by modulating p27Kip1 and p21WAF1/Cip1 protein levels. Moreover p27Kip1 is regulated post-translationally (via Skp2 regulation) and p21WAF1/Cip1 is regulated at transcriptional level. These results suggest that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in PaCa cells. Citation Format: Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. TET increases p27Kip1 protein stability and p21WAF1/Cip1 promoter activity to block deregulated cell cycle progression in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4305.

Cold-inducible RNA-binding protein (CIRP) induces translation of the cell-cycle inhibitor p27Kip1.

The CDK inhibitor p27Kip1 plays a central role in controlling cell proliferation and cell-cycle exit. p27Kip1 protein levels oscillate during cell-cycle progression and are regulated by mitogen or anti-proliferative signaling. The abundance of the protein is frequently determined by post-transcriptional mechanisms including ubiquitin-mediated proteolysis and translational control. Here, we report that the cold-inducible RNA-binding protein (CIRP) selectively binds to the 5′ untranslated region of the p27Kip1 mRNA. CIRP is induced, modified and relocalized in response to various stress stimuli and can regulate cell survival and cell proliferation particularly during stress. Binding of CIRP to the 5′UTR of the p27Kip1 mRNA significantly enhanced reporter translation. In cells exposed to mild hypothermia, the induction of CIRP correlated with increased translation of a p27Kip1 5′UTR reporter and with the accumulation of p27Kip1 protein. shRNA-mediated CIRP knockdown could prevent the induction of translation. We found that p27Kip1 is central for the decreased proliferation at lower temperature, since p27Kip1 KO mouse embryonic fibroblasts (MEFs) hardly increased their doubling time in hypothermic conditions, whereas wild-type MEFs significantly delayed proliferation in response to cold stress. This suggests that the CIRP-dependent p27Kip1 upregulation during mild hypothermia contributes to the cold shock-induced inhibition of cell proliferation.

Expression of p27Kip1, A Cell Cycle Repressor Protein with Dual Roles for Both Cancer Prevention and Promotion, Is Regulated Primarily at the Level of Unusual p27Kip1 mRNA—A Short Concept Proposal

The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.

Upregulation of miR-221 and -222 in response to increased extracellular signal-regulated kinases 1/2 activity exacerbates neointimal hyperplasia in diabetes mellitus

Background and aimsDiabetes is associated with accelerated arterial intimal thickening that contributes to the increased cardiovascular disease seen in this population. In healthy arteries, intimal thickening is inhibited by elevated levels of the cyclin-dependent kinase inhibitor, p27Kip1, and intimal thickening is promoted by activation of the mammalian Target of Rapamycin to promote degradation of p27Kip1 protein. Recently, we reported that two microRNAs, miR-221 and -222, which promote intimal thickening via down-regulation of mRNA encoding p27Kip1, are elevated in the arteries of diabetic patients. To determine if these miRNAs are critical to the increased intimal thickening under diabetic conditions, we examined the regulation of p27Kip1in a mouse model of diabetes. MethodsComparisons of p27Kip1 signaling in NONcNZO10 mice fed a diabetogenic versus control diet were performed using immunochemistry and real-time PCR. ResultsVascular smooth muscle cells and arteries of diabetic mice exhibited decreased levels of p27Kip1 that derived from destabilization of p27Kip1 mRNA in an extracellular signal response kinase-1/2 (ERK-1/2) dependent manner. The activity of ERK-1/2 is increased in the arteries of diabetic mice and promotes an increase in miR-221 and -222. Inhibition of miR-221 and -222 restores normal levels of p27Kip1 mRNA and protein in the arteries of diabetic mice and reduces intimal thickening following wire injury. ConclusionsThese data suggest diabetes is accompanied by increases in arterial miR-221 and -222 expression that promotes intimal thickening. Inhibition of the increased miR-221 and -222 may be efficacious in the prevention of the cardiovascular complications of diabetes.

Aberrant Level of Skp2 and p27KIP1 in Intraductal Proliferative Lesions is Associated with Tumorigenesis

ABSTRACTBreast cancer is one of the leading causes of cancer-related death in women worldwide. Here we aimed to examine the expression status of S-phase kinase-associated protein 2 (Skp2) and p27KIP1, and assess the significance of Skp2 plus p27KIP1 expression in patients with intraductal proliferative lesions, including ductal carcinoma in situ (DCIS) and atypical ductal hyperplasia (ADH). Skp2 and p27KIP1 mRNA levels in DCIS, ADH, flat epithelial atypia, and usual ductal hyperplasia (UDH) were evaluated by quantitative real-time reverse transcription polymerase chain reaction and protein expression was evaluated immunohistochemically in 60 fresh tissues and 120 paraffin-embedded tissues from patients with the four subtypes above. We found that the protein and mRNA level of Skp2 were significantly increased in DCIS and ADH as compared with that in UDH. In contrast, p27KIP1 protein and mRNA levels were reduced. Based on the above findings, abnormal levels of Skp2 and p27KIP1 have probably been involved in the pathogenesis of ADH and DCIS. Thus, Skp2 and p27KIP1 may serve as important diagnosis markers.

Peroxisome proliferator-activated receptor alpha deficiency impairs regulatory T cell functions: Possible application in the inhibition of melanoma tumor growth in mice.

Regulatory T (Treg) cells are important to induce and maintain immunological self-tolerance. Although the progress accomplished in understanding the functional mechanism of Treg cells, intracellular molecules that control the mechanisms of their suppressive capacity are still on investigation. The present study showed that peroxisome proliferator-activated receptor-alpha deficiency impaired the suppressive activity of Treg cells on CD4+CD25- and CD8+ T cell proliferation. In Treg cells, PPARα gene deletion also induced a decrease of migratory abilities, and downregulated the expression of chemokine receptors (CCR-4, CCR-8 and CXCR-4) and p27KIP1 mRNA. Treg cells from PPARα-/- mice also lost their anergic property. Since low Treg activity, as observed in PPARα-/- mice, is known to be associated with the inhibition of tumor growth, we inoculated these mice with B16 melanoma cells and assessed tumor proliferation. In PPARα-/- mice, cancer growth was significantly curtailed, and it was correlated with high expression of granzyme B and perforin mRNA in tumor bed. Degranulation of cytolytic molecules by CD8+ T cells, assessed by a perforin-release marker CD107a expression, was higher in PPARα-/- mice than that in wild-type mice. Tumor-infiltrating lymphocytes (TIL) in melanoma tumors in PPARα-/- mice exhibited high pro-inflammatory Th1 phenotype. Consistently, adoptive transfer into lymphopenic RAG2-/- mice of total PPARα-/-splenic T cells inhibited more the growth rate of B16 tumor than the wild type splenic T cells. Our findings suggest that PPARα deficiency, by diminishing Treg cell functions and upregulating pro-inflammatory T cell phenotype, exerts an invivo anti-cancer properties.

CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27(Kip1) mRNA translation.

The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3′ untranslated regions (3′UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27Kip1 by specifically targeting its 3′UTR, and competes with miR-221/222 binding at an overlapping site in the 3′UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27Kip1 3′UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27Kip1 mRNA. This leads to higher levels of p27Kip1 in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.

The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells.

Efficacy and mechanism of local delivery of rapamycin and rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on coronary restenosis of injury-stenosis model of minipigs

To determine whether intramural administration of rapamycin (RPM)-loaded polylactic-polyglycolic acid (PLGA) nanoparticles (NPs) can reduce intimal thickening and affect the mRNA expressions of matrix metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2 and p27(kipl) in a coronary injury-stenosis model of minipigs. Twenty eight minipigs were randomly separated into four groups: saline group (n=7), blank PLGA NPs group (5.0 mg/ml)(n=7), RPM group (1.0 mg/ml)(n=7), and RPM-PLGA NPs(5.0 mg/ml)group (n=7), respectively. Different treatments were intracoronary locally delivered via a Dispatch™ catheter for 10 minutes. Serial angiography was performed pre-and post-modeling 30 days and the percent stenosis degree was assessed. Hematoxylin-Eosin (HE) staining, Weigert's resorcin fuchsin staining and picric acid-sirius red staining were used for morphometric analysis. Immunohistochemistry was performed to assess the levels of proliferating cell nuclear antigen (PCNA), MMP-2, and TIMP-2 at early and late time points, respectively. The expression of p27(kip1) mRNA was detected by in situ hybridization staining. Data from 21 minipigs had been collected at the end of the experiment with 6, 4, 5, and 6 from the former mentioned 4 groups, respectively. For the instant injury index, there was no significant difference among the four groups. The percent stenosis degree of RPM-PLGA NPs group was significantly lower than that of the other three groups respectively (all P< 0.05). The neointima area, net external elastic lamina area to external elastic laminal area ratio, and proliferative index of RPM-PLGA NPs group were significantly less than those of the other three groups, with all the P values less than 0.05. The mean value of integral optical density of p27(kip1)mRNA expression of RPM-PLGA group was 0.35 ± 0.06, higher than that of blank PLGA NPs group (0.12 ± 0.05, P< 0.01), saline group (0.16 ± 0.03, P< 0.05), and RPM group (0.15 ± 0.03, P< 0.05), respectively. The MMP-2/TIMP-2 ratio and the positive expression index of PCNA in RPM-PLGA group were lower than that of the other groups (P< 0.05). Locally delivered rapamycin-loaded PLGA NPs significantly reduces MMP-2/TIMP-2 ratio and PCNA expression, increases p27(kip1) mRNA expression and significantly relieves percent stenosis degree and shows excellent acute procedural results in the minipig interventional coronary artery oversized balloon injury model. The results from minipig model further support that this approach could be a potential clinical procedure for vascular proliferative disease.

Pharmacokinetics and Biodistribution of Locked Nucleic Acid (LNA)-Mir-221 Inhibitor: A New Promising Anti-Myeloma Agent

miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression.We proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys.We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice.Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion.In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Abstract A186: Low dose paclitaxel treatment increase the stability of p27Kip1

Abstract The purpose of our study is to better understand how taxanes (paclitaxel, docetaxel) induce cell death. Taxanes play a critical role in combination chemotherapy for many types of solid tumors, especially in advanced disease states. These drugs bind to tubulin in interphase and mitotic microtubules altering their dynamics. This leads to G2/M arrest that is thought to contribute to taxane-induced apoptosis, although the exact mechanisms remain elusive. We found that after treating MDA-MB-231 breast cancer cells with low dose (10X IC50 = 40 nM) paclitaxel over 4-48 h, the cyclin-dependent kinase inhibitor p27Kip1 progressively increases more than 2-fold. p27Kip1 is an important regulator of cell cycle, found in both the nucleus and cytoplasm, and its accumulation is associated with G1 arrest in most cells. Its tumor suppressor activity is associated with its nuclear localization, where it forms complexes with Cdk2 and cyclin E (or other cyclins) preventing DNA replication and cell cycle advance. In the cytoplasm, p27Kip1 was shown to enhance cell motility. When p27Kip1 becomes phosphorylated on Thr 187 leading to its ubiquitination and degradation, the cell cycle can progress. We pretreated cells with 10 μM cycloheximide for 1 h to prevent translational peptide elongation and found when cells were subsequently treated with 40 nM paclitaxel for up to 4 hours, quantitative Western blotting showed that p27Kip1 levels steadily increased, indicating that paclitaxel treatment stabilized p27Kip1 protein. Quantitative real-time PCR demonstrated a parallel increase in p27Kip1 mRNA over 8 hours and then a gradual decline in mRNA over 24-48 h, while the protein remained stable over this time period. Since paclitaxel binds to microtubules, we wanted to know whether association of p27Kip1 with microtubules might play a role in its stabilization. We isolated the intact microtubule cytoskeleton from cells using a 2M glycerol buffer (100 mM Tris pH 6.8, 0.5% Nonidet P-40, 1 mM MgCl2 and 2 mM EGTA) at 37°C and found that 20% of the total intracellular p27Kip1 is associated with the microtubule pellet in control cells and 40 nM paclitaxel treatment for 48 h reduces this amount by about half, demonstrating that paclitaxel treatment leads to an increased amount of p27Kip1 in the non-microtubule fraction of the cell lysate. Because the tumor suppressor function of p27Kip1 is associated with its nuclear localization, we isolated nuclear and cytoplasmic fractions from MDA-MB-231 cell lysates and found a 2-fold change in p27Kip1 with 40 nM paclitaxel treatment for 48 h, compared to cells treated with DMSO. The 2-fold increase in p27kip1 was found entirely in the cytoplasmic fraction. Western blotting with an antibody that recognizes p27Kip1 phosphorylated at Thr 187 surprisingly showed a nearly 2-fold increase in phospho- p27Kip1 with paclitaxel treatment in the cytoplasmic fractions. In summary, paclitaxel treatment of MDA-MB-231 breast cancer cells leads to a 2-fold increase in total p27Kip1. This increase is primarily in the cytoplasmic fraction and is associated with an increase in phospho-The 187-p27Kip1. Our data together suggest that paclitaxel treatment may block ubiquitination of phospho -Thr 187-p27Kip1 or its proteosomal degradation. These novel observations are important for understanding the regulation of p27Kip1 and the mode of action for paclitaxel. Citation Format: Sharon Lobert, Mary E. Graichen. Low dose paclitaxel treatment increase the stability of p27Kip1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A186.

The regulatory loop of COMP1 and HNF-4-miR-150-p27 in various signaling pathways

MicroRNAs (miRNAs) are short regulatory RNAs that negatively modulate protein expression at the post-transcriptional level. Additionally, they have been associated with the pathogenesis of a number of types of cancer. In the current study, two target sites for miR-150 were determined within the 3′-untranslated region of p27Kip1 (hereafter referred to as p27) mRNA, and it was determined that ectopic overexpression of miR-150 led directly to p27 downregulation in cancer cells. These findings indicate that miR-150 may be a novel regulator of p27 expression. In the databases of the University of California, Santa Cruz (UCSC) and Match online, two common transcription factors were identified for miR-150 and p27: Cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4). Using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), it was determined that p27 is involved in pathways regulated by the target genes of miR-150. Therefore, these results suggest that there may be a regulatory loop between COMP1 and HNF-4-miR-150-p27. Additional functional studies are required to understand the molecular basis for the formation of this circuit loop, and provide an insight into the development of innovative therapies targeting specific tumor markers.

EZH2 is associated with malignant behavior in pancreatic IPMN via p27Kip1 downregulation.

BackgroundThe epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression.MethodsFifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of EZH2 and p27Kip1 mRNA were analyzed by real time PCR.ResultsIn immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma). EZH2 expression displayed a similar pattern (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma) with cell proliferative activity. EZH2 expression in malignant (CIS and invasive carcinoma) IPMNs was significantly higher than that in adenoma and borderline-atypia IPMNs. EZH2 expression level in IPMN lesions was positively correlated with the Ki-67 positive nuclear ratio (p<0.0001). EZH2-positive cells in malignant IPMN did not express p27Kip1. EZH2 mRNA expressions in malignant lesions were significantly higher than those in benign lesions (p<0.0001). In contrast, p27Kip1 mRNA in malignant lesions was significantly decreased compared to those in benign lesion (p<0.05), and there was an inverse correlation between EZH2 and p27Kip1 mRNA levels (p = 0.0109).ConclusionEZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27Kip1.