P27 Kip1 Degradation Research Articles

RNF4 promotes tumorigenesis, therapy resistance of cholangiocarcinoma and affects cell cycle by regulating the ubiquitination degradation of p27kip1 in the nucleus

Among the hallmarks of cholangiocarcinoma (CCA) progression and unresponsiveness to therapy is impaired ubiquitin-dependent degradation of nuclear tumor suppressor protein. In the previous stage, our research group found that as a key tumor suppressor, nuclear dysfunction of p27kip1 is closely related to chemotherapy resistance of CCA, but the specific mechanism is unclear. It was recently shown that p27kip1-driven tumors were strongly dependent on the SUMO pathway. RNF4, as the SUMO-targeted ubiquitin ligase (STUbL), identifies SUMOylated proteins as a substrate through sumo-interacting motifs (SIM) and causes its degradation via the ubiquitin proteasome pathway. Here we described that the expression of RNF4 was upregulated in CCA tissues and related to malignant features. Silencing RNF4 arrested human CCA cells at the G1 phase, which was associated with the upregulation of p27kip1 and the downregulation of its downstream cycle-related proteins. Silencing RNF4 inhibited cell proliferation and migration, increased cell apoptosis, and sensitized CCA cells to treatment of chemotherapeutic drugs in vitro. Immunofluorescence showed that p27kip1 and RNF4 were mainly co-located in the nucleus. Immunoprecipitation and Western blot showed that p27kip1 was a target protein for SUMOylation and high expression of RNF4 decreased the levels of nuclear p27kip1, enhanced the levels of ubiquitinated and SUMOylated p27kip1, indicating that RNF4 could regulate cell cycle progression via recognizing SUMOylated p27kip1 and facilitating its ubiquitination degradation. These data indicate that RNF4-mediated ubiquitination degradation of SUMOylated proteins is a novel regulatory mechanism of p27kip1 dysfunction and CCA tumorigenesis, which provides a potential option for therapeutic intervention of CCA.

Abstract 668: A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo

Abstract Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 668.

Anti-tumor effects of Skp2 inhibitor AAA-237 on NSCLC by arresting cell cycle at G0/G1 phase and inducing senescence

Lung cancer is by far the leading cause of cancer death worldwide, and 85% of patients are diagnosed with non-small cell lung cancer (NSCLC), which is still very difficult to treat. Skp2 functions as an oncogene that participates in processes of many cancers. Here, we report a novel Skp2 inhibitor AAA-237 that binds to Skp2 protein and inhibits the proliferation of the NSCLC cells. We further investigated the anti-NSCLC mechanism of AAA-237 and found that it arrested the cell cycle at the G0/G1 phase by targeting Skp2 to reduce the degradation of p21Cip1 and p27Kip1 or by transcriptionally activating FOXO1 to increase the mRNA expression of p21Cip1 and p27Kip1. More importantly, we found that treatment of a high concentration AAA-237 could induce apoptosis of NSCLC cells and treatment of a low AAA-237 concentration for a longer time could induce senescence of NSCLC cells. Similar results were found in nude mice xenografted with A549 cells. AAA-237 inhibited tumor growth by inducing apoptosis and senescence in a dose-dependent manner. Considering these results, we propose that AAA-237 could be a promising therapeutic drug for treating patients with NSCLC.

Characterization of SCF Ligase Inhibition by Tumor Suppressor HINT1

Skp, Cullin, F-box containing (SCF) ubiquitin E3 ligases are part of the largest family of E3 ligases. They ubiquitylate key proteins in many biological pathways. in recent years, in vivo studies have shown that the protein Histidine triad nucleotide-binding protein 1 (HINT1) inhibits the ubiquitylation activity of various SCF ligases that regulate the NF-κB signaling pathway and cell cycle progression by promoting the degradation of substratesIκBα and p27Kip1, respectively. However, the mechanism of interaction between HINT1 and SCF ligases and how HINT1 causes their inhibition is not understood. Many questions remain unanswered such as the specific binding partner of HINT1 within the SCF complex and how HINT1 is able to inhibit the activity of unique SCF ligases. in total, the ∼173 kDa SCF ligase complex consists of a scaffolding protein, Cullin1 (90 kDa), that utilizes Rbx1 (12 kDa) to recruit E2 ubiquitin conjugating enzymes (e.g. UBE2D2) and substrates through the adaptor and substrate receptor proteins, Skp1 (17 kDa) and β-TrCP1 (54 kDa) respectively, to promote substrate ubiquitylation. Through co-elution in gel-filtration chromatography, I have assembled the SCF E3 ligase that ubiquitylates IκBα and have demonstrated that HINT1 (28 kDa) binds to this ligase. I have performed biochemical experiments to measure ubiquitylation combined with HDX-MS and other biophysical experiments to characterize the binding affinity and the specific binding interface of HINT1 on the SCF E3 ligase. The results shed light on the molecular mechanism of inhibition of SCF E3 ligases by HINT1.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

VCP/p97 targets the nuclear export and degradation of p27Kip1during G1 to S phase transition

One of the critical regulatory mechanisms for cell cycle progression is the timely degradation of CDK inhibitors, including p21Cip1 and p27Kip1 . VCP/p97, an AAA-ATPase, is reported to be overexpressed in many types of cancers. Here, we found that treatment of MCF-7 human breast cancer cells with DBeQ, a VCP inhibitor, or VCP knockdown in MCF-7 cells arrested cells at G1 phase, accompanied with the blockage of both p21 and p27 degradation. Whereas, double knockdown of p21 and p27 in MCF-7 cells rendered cells refractory to DBeQ-induced G1 arrest. Moreover, inhibition or knockdown of VCP or UFD1, one of VCP's co-factors, in MCF-7, NIH3T3, or HEK293T cells blocked the nuclear export of p27 during earlier G1 phase after mitogen stimulation. We also identified the nuclear localization sequence (NLS) of VCP, and found that adding back wild-type VCP, not the NLS-deleted VCP mutant, restored the nuclear export and degradation of p27 in VCP knockout MCF-7 cells. Importantly, we found that VCP inhibition sensitized breast cancer cells to the treatment of several anticancer therapeutics both in vitro and in vivo. Taken together, our study not only uncovers the mechanisms underlying VCP-mediated cell proliferation control but also provides potential therapeutic option for cancer treatment.

Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential Mechanism.

Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

Two Pathways of p27Kip1 Degradation Are Required for Murine Lymphoma Driven by Myc and EBV Latent Membrane Protein 2A.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and MYC transgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressing MYC (λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1 is present at significantly lower levels in LMP2A/λ-MYC mice due to increased posttranslational degradation. P27Kip1 degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10) or in the nucleus via the SCFSkp2 complex, which depends on Cks1. We previously demonstrated an S10A knock-in of p27Kip1 (p27S10A/S10A) that prevented S10 phosphorylation failed to significantly delay tumor onset in LMP2A/λ-MYC mice. We also previously demonstrated that a Cks1 knockout partially delayed tumor onset in LMP2A/λ-MYC mice, but onset was still significantly faster than that in λ-MYC mice. Here, we have combined both genetic manipulations in what we call p27Super mice. LMP2A/λ-MYC/p27Super mice and λ-MYC/p27Super mice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Super mice was later than that in λ-MYC mice and not significantly different from that in λ-MYC/p27Super mice. The p27Super genotype also normalized G1-S-phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYC mice. Our results reveal both major pathways of p27Kip1 degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.IMPORTANCE BL is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain EBV, and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression of MYC Mice engineered to express EBV LMP2A along with MYC (LMP2A/λ-MYC mice) develop tumors much more quickly than mice only expressing MYC (λ-MYC mice). Blocking degradation of the cell cycle inhibitor protein p27Kip1 in LMP2A/λ-MYC mice causes tumors to develop later than in λ-MYC mice, showing that p27Kip1 degradation may play a larger role in EBV-positive BL than EBV-negative BL. Furthermore, our studies suggest the cell cycle is an attractive target as a treatment option for LMP2A-positive cancers in humans.

A BAP31 intrabody induces gastric cancer cell death by inhibiting p27kip1 proteasome degradation.

B-cell receptor-associated protein 31 (BAP31) is a ubiquitously expressed endoplasmic reticulum (ER) membrane protein that has been found to be overexpressed in gastric intestinal-type adenocarcinoma. We first studied the relationship of BAP31 with 84 kinds of tumor-associated antigens and found that BAP31 can specifically interact with and regulate the proteasome degradation of the cyclin kinase inhibitor p27kip1 , which is one of the most frequently dysregulated tumor suppressor proteins in human cancers. Therefore, we screened antibodies against BAP31 from a human VH single-domain antibody library and expressed the antibodies intracellularly. It was found that one of the intrabodies (VH-D1) specifically inhibited p27kip1 proteasome degradation, possibly by blocking the combination of BAP31 with p27kip1 . VH-D1 displayed therapeutic effects, as it was able to reduce the growth of human gastric cancer (GC) cell xenografts in nude mice. This effect was due to inhibition of the proliferation and subsequent activation of caspase-dependent apoptosis. Thus, BAP31 is a potential target for the suppression of GC via an intrabody-based approach.

Outcome of Multiple Myeloma with Chromosome 1q Gain and 1p Deletion after High-Dose Therapy and Autologous Hematopoietic Stem Cell Transplantation

Background and AimThe gain/amplification CKS1B gene at chromosome region 1q21 (1q+) is one of the most common genetic aberrations in multiple myeloma (MM). CKS1Bamplification and overexpression contribute to increased p27Kip1 degradation, cell cycle upregulation and poor outcomes in MM patients. Amplification of CKS1B is frequently associated with the deletion of CDKN2C gene at chromosome region 1p32 (1p-). Deletion of CDKN2C, a tumor suppressor gene, leads to an increased proliferative rate of plasma cells in MM patients and is also associated with inferior outcomes. There are limited data on the impact of 1q+/1p- on the outcomes after high-dose therapy and autologous stem cell transplantation (auto-HCT).MethodsIn this retrospective study, we evaluated outcomes of patients with 1q+ and/or 1p- after high-dose therapy and auto-HCT. From 2007 to 2015, 1365 MM patients underwent high-dose therapy and auto-HCT at our institution. We identified 100 patients with 1q+ and/or 1p- by fluorescent in situ hybridization (FISH) from that cohort. The cut off value used for gain/amplification of a 1q21/CKS1B was 7.9%; gain/amplification of two 1q21/CKS1B was 4.4%; and deletion of a 1p32.3/CDKN2C was 6.8%. A control group (N=287) with diploid cytogenetics and normal FISH panel with no high risk abnormalities was selected from the same cohort. From the above two cohorts, using a 1:1 propensity score-matched analysis without replacement, we were able to identify a matched control for 85 patients with 1q+/1p-.ResultsBaseline characteristics of the 1q+/1p- and control groups are summarized in table 1. Sixty-seven (79%), 4 (5%) and 14 (16%) patients had 1q+, 1p- or both 1q+ and 1p-, respectively. There was no significant difference in induction therapy, preparative regimen, or maintenance therapy between the two groups. The median follow-up time for all patients was 29.2 months (range: 0.29 -84.96). The cumulative incidence of 100-day nonrelapse mortality was 1.1% and 0% for the 1q+/1p- and the control groups, respectively. Forty-two patients (51%) in the 1q+/1p- group achieved a CR compared to 40 patients (49%) in the control group (P=0.6). VGPR rates in the 1q+/1p- and controls groups were 47% and 53% (P=0.5) respectively. The overall response rate for the 1q+/1p-and controls group was 96% and 100%, respectively (P=0.3). Median PFS for the 1q+/1p- and the controls groups were 26.5 months and 38.8 months, respectively. The estimated 3-year PFS for the 1q+/1p- and the control groups were 41% and 56%, respectively (HR 2.2, CI 1.18-4.16, P=0.01) (Figure 1A). The 3-year OS for the 1q+/1p- and the control groups were 79% and 86%, respectively (HR 1.5, CI 0.61-4.05, P=0.34) (Figure 1B).Conclusion1q+/1p- abnormalities with amplification CKS1B and deletion of CDKN2C genes were seen in approximately 7% of MM patients undergoing auto-HCT between 2007 and 2015. They were associated with a shorter PFS when compared to a propensity matched group of patients with diploid cytogenetics and normal FISH panel. Patients with 1q+/1p- may need more aggressive therapeutic approaches. DisclosuresThomas:Amgen Inc: Research Funding; Bristol Myers Squibb Inc.: Research Funding; Array Pharma: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding. Lee:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Patel:Poseida Therapeutics, Inc.: Research Funding; Takeda: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Shpall:Affirmed GmbH: Research Funding. Orlowski:Bristol-Myers Squibb: Consultancy; Kite Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding; Sanofi-Aventis: Consultancy; Janssen: Consultancy; Spectrum Pharma: Research Funding; BioTheryX: Research Funding; Takeda: Consultancy. Champlin:Otsuka: Research Funding; Sanofi: Research Funding.

Tsantan Sumtang Alleviates Chronic Hypoxia-Induced Pulmonary Hypertension by Inhibiting Proliferation of Pulmonary Vascular Cells.

Hypoxia-induced pulmonary hypertension (HPH) is a severe condition associated with significant morbidity and mortality in people living at high altitude. Tsantan Sumtang, a traditional Tibetan medicine, has been routinely used for the treatment of cardiopyretic disease, as well as stenocardia. Interestingly, our previous research found that Tsantan Sumtang improved HPH in rats maintaining in a hypobaric chamber. We performed a series of experiments to test the indexes of vasoconstriction and vascular remodeling, the key pathophysiological characteristics of HPH. Our results showed that Tsantan Sumtang relaxed noradrenaline (NE)-precontracted rat pulmonary artery rings in a concentration-dependent manner in vitro. The PGI2-cAMP (prostaglandin I2-cyclic adenosine monophosphate) pathway, NO-cGMP (nitric oxide-cyclic guanosine monophosphate) pathway, and the opening of K+ channels (inward rectifier K+ channels, large conductance Ca2+-activated K+ channels, and voltage-dependent K+ channels) might play major roles in the vasorelaxation effect. In vivo, the administration of Tsantan Sumtang resulted in a substantial decrease in the rat mean pulmonary artery pressure (mPAP) and the right ventricular hypertrophy index (RVHI). The reduction of thickness of small pulmonary arterial wall and the WT% (the ratio of the vascular wall thickness to the vascular diameter) were observed. The smooth muscle muscularization of the arterials was alleviated by Tsantan Sumtang treatment at the same time. Tsantan Sumtang also reduced remodeling of pulmonary arterioles by suppressing the expression of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), cyclin D1, and cyclin-dependent kinase 4 (CDK4) through inhibition of p27Kip1 degradation. Therefore, Tsantan Sumtang could be applied as a preventative medication for HPH, which would be a new use for this traditional medicine.

Corrigendum to "Long non-coding RNA LINC-01572:28 inhibits granulosa cell growth via a decrease in p27 (Kip1) degradation in patients with polycystic ovary syndrome" [EBioMedicine 36 (2018) 526-538

Corrigendum to "Long non-coding RNA LINC-01572:28 inhibits granulosa cell growth via a decrease in p27 (Kip1) degradation in patients with polycystic ovary syndrome" [EBioMedicine 36 (2018) 526-538

Long non-coding RNA LINC-01572:28 inhibits granulosa cell growth via a decrease in p27 (Kip1) degradation in patients with polycystic ovary syndrome.

BackgroundDisordered folliculogenesis is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear.MethodsLong non-coding RNA (lncRNA) expression in luteinized granulosa cells (hLGCs) derived from women with and without PCOS were analyzed using microarray and qRT-PCR. Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 siRNA. Protein expression analysis, rescue assays, and RNA immunoprecipitation (RIP) were used to confirm the LINC-01572:28 substrate.FindingsLINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the hLGCs of women with PCOS. LINC-01572:28 expression was positively correlated with basal testosterone levels. Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition, which were partially reversed by siRNA-mediated p27 knockdown.InterpretationOur findings, therefore, suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding.

Long Non-Coding RNA LINC-01572:28 Inhibits Granulosa Cell Growth Via a Decrease in p27 (Kip1) Degradation in Patients with Polycystic Ovary Syndrome

Disordered folliculogenesis is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear. Long non-coding RNA (lncRNA) expression in luteinized granulosa cells (hLGCs) derived from women with and without PCOS were analyzed using microarray and qRT-PCR. Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 siRNA. Protein expression analysis, rescue assays, and RNA immunoprecipitation (RIP) were used to confirm the LINC-01572:28 substrate. LINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the hLGCs of women with PCOS. LINC-01572:28 expression was positively correlated with basal testosterone levels. Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition, which were partially reversed by siRNA-mediated p27 knockdown. Our findings, therefore, suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding. Funding: This research was supported by grants from the National Key Research and Development Program of China(2017YFC1001002, the National Natural Science Foundation (81490743, 81671413 and 81671414), National Institutes of Health (1R01HD085527), Shanghai Municipal Education Commission--Gaofeng Clinical Medicine (20152510) and Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics (17DZ2271100). Declaration of Interests: Authors have no conflicts of interest to declare. Ethical Approval Statement: The study was approved by the ART Ethnics committee of Ren Ji hospital, school of medicine, shanghai Jiao Tong university (number: 2017041411), and informed consent was obtained from all participants.

Naringenin is an inhibitor of T cell effector functions

Selective inhibition of T cells has been implied to prevent and/or treat autoimmune and inflammatory diseases. Some food compounds that have such immune-modulating functions may serve as nutritional approach to this purpose. In this study, we chose naringenin, a citrus fruits-derived compound with antiinflammatory property, to test this possibility. In this in vitro study, we stimulated mouse T cells with anti-CD3/CD28 (polyclonal TCR activation) or autoantigen MOG35–55 in the presence of naringenin. We found that naringenin dose-dependently suppressed anti-CD3/CD28 and MOG35–55-induced T cell proliferation, production of T cell cytokines IFN-γ, IL-17, IL-6 and TNF-α. We further showed that inhibited T cell proliferation was associated with T cell cycle arrest at G0/G1 phase, which was in turn related to delayed degradation of the cyclin-dependent kinase inhibitor p27kip1 and the down-regulation of retinoblastoma protein phosphorylation in activated T cells. Finally, it was revealed that all these T cell-suppressive effects might be related to naringenin's interference with IL-2/IL-2R-mediated signaling pathway and STAT5 phosphorylation in activated T cells. Our results confirmed T cell-suppressive activity of naringenin previously reported by us and others, but for the first time, it was shown that the working mechanism may involve its ability to modulate cell cycle progression, cell cycle-related proteins and IL-2/IL-2R signaling pathway. Together, these results further support proposed potential of naringenin being a preventive/therapeutic agent in T-cell-mediated autoimmune inflammatory disorders.

Tetrandrine inhibits deregulated cell cycle in pancreatic cancer cells: Differential regulation of p21Cip1/Waf1, p27Kip1 and cyclin D1

Current therapies in Pancreatic Cancer (PaCa) are ineffective due to deregulated cell cycle driven by landscape mutations. In this study, we show for the first time that tetrandrine (TET) inhibits proliferation of the PaCa cells and inhibits PaCa tumor growth. TET inhibits cell cycle transition at G1/S boundary. TET increased levels of p21Cip1/Waf1 and p27Kip1, had no effect on the levels of CDK4/6 proteins and decreased the levels of cyclin D1 and pRb proteins. TET resulted in changes in mRNA levels of cyclin D1 and p21Cip1/Waf1 but had no effect on the mRNA of p27Kip1. We show, for the first time in any system, that TET treatment downregulated Skp2, E3 ligase specific for degradation of p27Kip1 during the cell cycle. Taken together our results show, that TET indirectly impairs the activities of CDK4/6 to halt deregulated cell cycle and inhibit PaCa tumor growth. These results suggest that TET may serve as a novel agent for treatment of PaCa, for which there is no effective cure to date.

Bioactive fraction of Rhodiola algida against chronic hypoxia-induced pulmonary arterial hypertension and its anti-proliferation mechanism in rats

Ethnopharmacological relevanceRhodiola algida var. tangutica (Maxim.) S.H. Fu is a perennial plant of the Crassulaceae family that grows in the mountainous regions of Asia. The rhizome and roots of this plant have been long used as Tibetan folk medicine for preventing high latitude sickness. Aim of the studyThe aim of this study was to determine the effect of bioactive fraction from R. algida (ACRT) on chronic hypoxia-induced pulmonary arterial hypertension (HPAH) and to understand the possible mechanism of its pharmacodynamic actions. Materials and methodsMale Sprague-Dawley rats were separated into five groups: control group, hypoxia group, and hypoxia+ACRT groups (62.5, 125, and 250mg/kg/day of ACRT). The chronic hypoxic environment was created in a hypobaric chamber by adjusting the inner pressure and oxygen content for 4 weeks. After 4 weeks, major physiological parameters of pulmonary arterial hypertension such as mPAP, right ventricle index (RV/LV+S, RVHI), hematocrit (Hct) levels and the medial vessel thickness (wt%) were measured. Protein and mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, p27Kip1 and cyclin-dependent kinase 4 (CDK4)) were detected by western blotting and real time PCR respectively. Chemical profile of ACRT was revealed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS). ResultsThe results showed that a successful HPAH rat model was established in a hypobaric chamber for 4 weeks, as indicated by the significant increase in mPAP, RV/LV+S, RV/BW and wt%. Compared with the normal group, administration of ACRT reduced mPAP, right ventricular hypertrophy, pulmonary small artery wall thickness, and damage in ultrastructure induced by hypoxia in rats. PCNA, cyclin D1, and CDK4 expression was reduced (p<0.05), and p27Kip1 expression increased (p<0.05) in hypoxia+ACRT groups compared to hypoxia. 38 constituents in bioactive fraction were identified by UHPLC-Q-TOF-MS/MS. ConclusionOur results suggest that ACRT could alleviate chronic hypoxia-induced pulmonary arterial hypertension. And its anti-proliferation mechanism in rats based on decreasing PCNA, cyclin D1, CDK4 expression level and inhibiting p27Kip1 degradation.

EBV latent membrane protein 2A orchestrates p27kip1 degradation via Cks1 to accelerate MYC-driven lymphoma in mice

AbstractEpstein-Barr virus (EBV) establishes lifelong infection in B lymphocytes of most human hosts and is associated with several B lymphomas. During latent infection, EBV encodes latent membrane protein 2A (LMP2A) to promote the survival of B cells by mimicking host B-cell receptor signaling. By studying the roles of LMP2A during lymphoma development in vivo, we found that LMP2A mediates rapid MYC-driven lymphoma onset by allowing B cells to bypass MYC-induced apoptosis mediated by the p53 pathway in our transgenic mouse model. However, the mechanisms used by LMP2A to facilitate transformation remain elusive. In this study, we demonstrate a key role of LMP2A in promoting hyperproliferation of B cells by enhancing MYC expression and MYC-dependent degradation of the p27kip1 tumor suppressor. Loss of the adaptor protein cyclin-dependent kinase regulatory subunit 1 (Cks1), a cofactor of the SCFSkp2 ubiquitin ligase complex and a downstream target of MYC, increases p27kip1 expression during a premalignant stage. In mice that express LMP2A, Cks1 deficiency reduces spleen weights, restores B-cell follicle formation, impedes cell cycle progression of pretumor B cells, and eventually prolongs MYC-driven tumor onset. This study demonstrates that LMP2A uses the role of MYC in the cell cycle, particularly in the p27kip1 degradation process, to accelerate lymphomagenesis in vivo. Thus, our results reveal a novel mechanism of EBV in diverting the functions of MYC in malignant transformation and provide a rationale for targeting EBV's roles in cell cycle modulation.

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation.

The molecular pathogenesis of human lung cancer has not been completely clarified. Here, we reported that UBE2L3, a member of the ubiquitin-conjugating enzymes (E2s), were overexpressed in non-small-cell lung cancer (NSCLC) tissues compared with the non-tumor tissues. High expression of UBE2L3 was correlated with advanced tumor stage and adverse outcomes. Knockdown of UBE2L3 inhibited NSCLC cell growth while ectopic expression of UBE2L3 promoted NSCLC cell growth in a cell cycle dependent manner. The results of subcutaneous tumor xenograft studies revealed that knockdown of UBE2L3 attenuated the in vivo tumor growth. Mechanistically, we observed that UBE2L3 could interact with F-box protein Skp2, a member of the SCF (Skp2) ubiquitin ligase complex, and thus promoted the ubiquitination and proteasomal degradation of p27kip1. Furthermore, NSCLC cases with high level of UBE2L3 and low level of p27kip1 had worst prognosis, suggesting that combination of UBE2L3 and p27kip1 is a more powerful prognostic marker for NSCLC patients. Taken together, the current study presented a novel marker for predicting prognosis and a potential therapeutic target for NSCLC patients.