P27 Kip1 Expression Research Articles

P.258 Depression and suicidal ideation in treatment resistant depression patients in Latin America: final results from the multicentre, prospective, observational TRAL study

Somatostatin (SST) mediates cytostatic and pro-apoptotic effects through five somatostatin receptors (SSTR1-5). The modest clinical benefits of SST analogs in cancers of different origin such as breast cancer are attributed to diminished SSTRs expression at tumor sites. In the present study, SSTR3 was overexpressed in MCF-7 and MDA-MB-231, and analyzed for downstream signaling molecules associated with cytostatic and cytotoxic effect. Cells overexpressing SSTR3 displayed inhibition of EGF induced proliferation and enhanced antiproliferative effect of SSTR3-specific agonist in comparison to non-transfected cells. SSTR3 overexpression in MCF-7 cells (R3-MCF-7) constitutively enhanced TUNEL staining, PARP-1 and p27Kip1 expression suggesting apoptosis and cell-cycle arrest. Conversely, R3-MB-231 cells with SSTR3 overexpression exerted cytostatic and were devoid of any cytotoxic effects. The expression of PTP-1C and the status of ERK1/2, p38 and PI3K phosphorylation was modulated in a cell-specific manner. These findings provide new insights in understanding the antiproliferative role of SSTR3 in breast tumor biology.

Transmembrane and Immunoglobulin Domain Containing 1, a Putative Tumor Suppressor, Induces G2/M Cell Cycle Checkpoint Arrest in Colon Cancer Cells

Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target.

Increased Expression of NPM1 Suppresses p27Kip1 Function in Cancer Cells.

Simple SummaryCancer malignancy frequently correlates with a low expression of p27Kip1, a major cyclin-dependent kinase inhibitor, and the p27 protein level has been reportedly responsible for its antiproliferative function. However, we found the function of overexpressed p27 is suppressed in some cancer cells, suggesting that p27 function is also regulated independently of its protein level. The aim of this study was to clarify this unknown p27 regulatory mechanism and its impact on cancer proliferation. We isolated nucleophosmin isoform 1 (NPM1), which is highly expressed in variety of cancers, as a novel p27-interacting protein. Overexpressing NPM1 in normal cells suppressed and silencing NPM1 in cancer cells rescued the p27 function, respectively, in vitro. Moreover, NPM1 silencing and p27 induction in cancer cells significantly suppressed their proliferation in mouse xenografts. Our findings reveal that NPM1 is a novel p27 functional suppressor and a potential anti-cancer target, especially in cancers with normal p27 expression.p27Kip1, a major cyclin-dependent kinase inhibitor, is frequently expressed at low levels in cancers, which correlates with their malignancy. However, in this study, we found a qualitative suppression of p27 overexpressed in some cancer cells. By proteomic screening for factors interacting with p27, we identified nucleophosmin isoform 1 (NPM1) as a novel p27-interacting factor and observed that NPM1 protein was expressed at high levels in some cancer cells. NPM1 overexpression in normal cells suppressed p27 function, and conversely, NPM1 knockdown in cancer cells restored the function in vitro. Furthermore, the tumors derived from cancer cells carrying the combination of p27 overexpression and NPM1 knockdown constructs showed significant suppression of growth as compared with those carrying other combinations in mouse xenograft models. These results strongly suggest that increased expression of NPM1 qualitatively suppresses p27 function in cancer cells.

Sauchinone Protects Renal Mesangial Cell Dysfunction against Angiotensin II by Improving Renal Fibrosis and Inflammation.

Abnormal and excessive growth of mesangial cells is important in the pathophysiologic processes of diabetes-associated interstitial fibrosis and glomerulosclerosis, leading to diabetic nephropathy, which eventually turns into end-stage renal disease. Sauchinone, a biologically-active lignan isolated from aerial parts of Saururus chinensis, has anti-inflammatory and anti-viral activities effects on various cell types. However, there are no studies reporting the effects of sauchinone on diabetic nephropathy. The present study aims to investigate the role of sauchinone in mesangial cell proliferation and fibrosis induced by angiotensin II, as well as the underlying mechanisms of these processes. Human renal mesangial cells were induced by angiotensin II (AngII, 10 μM) in the presence or absence of sauchinone (0.1–1 μM) and incubated for 48 h. In this study, we found that AngII induced mesangial cell proliferation, while treatment with sauchinone inhibited the cell proliferation in a dose-dependent manner. Pre-treatment with sauchinone induced down-regulation of cyclins/CDKs and up-regulation of CDK inhibitor, p21, and p27kip1 expression. In addition, AngII-enhanced expression of fibrosis biomarkers such as fibronectin, collagen IV, and connective tissue growth factor (CTGF), which was markedly attenuated by sauchinone. Sauchinone also decreased AngII-induced TGF-β1 and Smad-2, Smad-3, and Smad-4 expression. This study further revealed that sauchinone ameliorated AngII-induced mesangial inflammation through disturbing activation of inflammatory factors, and NLRP3 inflammasome, which is composed of the NLRP3 protein, procaspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC). Moreover, pretreatment of sauchinone inhibited NF-κB translocation and ROS production in AngII-exposed mesangial cells. These data suggest that sauchinone has a protective effect on renal proliferation, fibrosis and inflammation. Therefore, sauchinone might be a potential pharmacological agent in prevention of AngII-induced renal damage leading to diabetic nephropathy.

USP7 promotes proliferation of papillary thyroid carcinoma cells through TBX3-mediated p57KIP2 repression

Ubiquitin-specific protease 7 (USP7/HAUSP) is known to regulate multiple cellular phenomena, including cell cycle progression and proliferation, and is involved in binding and stabilizing specific target proteins through deubiquitylation. However, the detailed role of USP7 in papillary thyroid carcinoma (PTC) remains to be investigated. In this study, our results showed that USP7 was upregulated in PTC tissues compared with adjacent nontumour tissues. Consistently, a series of gain/loss functional assays in vivo and in vitro demonstrated the role of USP7 in promoting PTC cell proliferation. Furthermore, we showed that there was a negative correlation between USP7 and the CDK inhibitor p57KIP2 expression in PTC tissues and that USP7 facilitated PTC cell proliferation by inhibiting p57KIP2. Mechanistically, USP7 inhibited p57KIP2 expression by modulating TBX3, directly binding to TBX3, and decreasing its ubiquitination and degradation. Our findings demonstrated that USP7 played a critical oncogenic role in PTC tumorigenesis, suggesting that USP7 might act as a prognostic and therapeutic target for PTC progression.

MicroRNA-150 suppresses p27Kip1 expression and promotes cell proliferation in HeLa human cervical cancer cells.

MicroRNAs (miRNAs) exert critical roles in the majority of biological and pathological processes. Recent studies have associated miR-150 with a number of different cancer types. However, little is known about miR-150 targets in cervical cancer. In the present study, the HeLa human cervical cancer cell line was transfected with hsa-miR-150-5p mimics, hsa-miR-150-5p inhibitors or miRNA controls. miR-150 was predicted to bind the 3′untranslated region (3′UTR) of the CDKN1B gene, which encodes the cyclin-dependent kinase inhibitor 1B (p27Kip1). The direct binding between miR-150 and the 3′UTR of CDKN1B was confirmed using dual-luciferase reporter assays. The effects of miR-150 on CDKN1B mRNA expression, p27Kip1 protein expression, cell cycle and cell proliferation were determined using reverse-transcription quantitative PCR, western blot analysis, flow cytometry and WST-8 assays, respectively. miR-150 was demonstrated to directly target the 3′UTR of CDKN1B in transfected HeLa cells. The expression of CDKN1B mRNA and p27Kip1 protein was reduced by miR-150 mimics, and increased by miR-150 inhibitors. Moreover, the overexpression of miR-150 promoted cell cycle progression from the G0/G1 to the S phase and led to a significant increase in HeLa cell proliferation. The results of the present study indicated that miR-150 promotes HeLa cell cycle progression and proliferation via the suppression of p27Kip1 expression.

AKT/GSK-3β/β-catenin signaling pathway participates in erythropoietin-promoted glioma proliferation.

Although erythropoietin (EPO) has been proven to significantly promote the proliferation of cancer cells, the mechanism for promoting glioma proliferation is poorly understood. Here, we examined the functional role of the AKT/GSK-3β/β-catenin signaling pathway in the EPO-mediated proliferation of glioma. The distribution of EPO and Ki-67 among clinical samples with different WHO grades was plotted by Immunological Histological Chemistry analysis. U87 and U251 glioma cell lines were treated with short hairpin RNA targeting (shEPO), recombinant human erythropoietin (rhEPO) and/or AKT-specific inhibitor (MK-2206). The changes in phosphorylated AKT, nuclear β-catenin, cyclin D1 and p27kip1 expression were detected.Cell cycle distributions and glioma proliferation in vitro and in vivo were analyzed. The expression level of EPO was significantly elevated with the increase of WHO grade and Ki67 in clinical glioma specimens. In vitro, knockdown of endogenous EPO in U87 and U251 cells effectively block the phosphorylation of AKT and GSK-3β and the expression of nuclear β-catenin. shEPO treatment also significantly decreased the expression of cyclin D1 and increased the expression of p27kip1. The cell cycle transition then slowed down and the proliferation of glioma cells or mouse xenograft tumors both decreased. Treatment of cells or tumors with extra rhEPO reversed the above biological effects mediated by shEPO. rhEPO-induced activation of the AKT/GSK-3β/β-catenin pathway and proliferation were abolished by MK-2206. Our study identified the AKT/GSK-3β/β-catenin axis as a critical mediator of EPO-induced glioma proliferation and further provided a clinically significant dimension to the biology of EPO.

The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast

BackgroundThis study aimed to investigate the regulation of CpG oligodeoxynucleotides (ODNs) on proliferation and osteogenic differentiation of MC3T3 cells.MethodsThe laser co-focusing and flow cytometry assay were employed to detect cell uptake of CpG ODN 2006. Twelve ODNs were sythesized, and their effects on proliferation and differentiation were detected by MTT and alkaline phosphatase (ALP) activity assay. Flow cytometry assay was used to examine the regulation of CpG ODN on cell cycle. Quantitative real-time PCR (qRT-PCR) and western blot were used to evaluate the regulation of CpG ODN on mRNA and protein expression of osteogenic differentiation genes.ResultsThe phosphorothioate CpG ODN 2006 could efficiently enter the MC3T3 cells in 1 h and locate in the cytoplasm. The MTT assay demonstrated CpG ODNs could promote MC3T3 cell proliferation and differentiation in the early stage, and gradually attenuated along with the increase of treating time, except for BW001 and FC001. qRT-PCR assay demonstrated that all the 12 CpG ODNs could promote the relative expression level of osteogenic differentiated genes, SP7 and OCN. In addition, western blot analysis suggested the CpG ODNs of BW001 and FC001 could increase the protein expression of P27Kip1 and Runx2 and decrease the protein expression of cyclin D1.ConclusionThe selected CpGODNs may be a potential gene therapy for bone regeneration of periodontitis.

Abstract 5815: Sox2 elevation downregulates a broad spectrum of cell cycle machinery and induces a reversible state of tumor growth arrest

Abstract Purpose: The purpose of these studies was to understand how elevated SOX2 affects tumor cell proliferation. The stem cell transcription factor SOX2 has been implicated in the progression and chemoresistance of ~20 different tumor types. Importantly, several studies have demonstrated that the effects of SOX2 on proliferation are dependent on its dosage. Significantly, elevated SOX2 expression is associated with restricted cell proliferation during development and tumor progression. SOX2high, quiescent tumor cells present a significant clinical challenge as these cells are largely resistant to conventional chemotherapies and thus, provide a reservoir to drive disease recurrence. Therefore, understanding the mechanisms through which elevated SOX2 inhibits tumor cell proliferation will provide insights into the biology of drug-resistant, quiescent tumor cells. Methods: A diverse group of tumor cell lines representative of medulloblastoma (MB), prostate cancer (PCa), neuroblastoma (NB), and pancreatic ductal adenocarcinoma (PDAC) were engineered to express SOX2 from a doxycycline (Dox)-inducible promoter. The effect of SOX2 elevation on proliferation was examined both in vitro and in vivo. Cell cycle analysis was performed by flow cytometry. Kinetics of tumor growth were determined by the subcutaneous engraftment of SOX2-inducible tumor cells in nude mice. SOX2 expression was induced in vivo by addition of Dox to the water of the mice. Results: Elevating SOX2 in MB, PCa, NB, and PDAC cells inhibited the proliferation of each cell line in vitro. Intriguingly, SOX2 elevation in six different tumor cell lines did not significantly alter their cell cycle distribution, despite strongly inhibiting proliferation. In agreement with these results, western blot analysis of whole cell extracts determined that elevating SOX2 leads to the downregulation of a broad spectrum of cyclins and cyclin-dependent kinases (CDKs) governing progression through multiple phases of the cell cycle. Additionally, SOX2 elevation increased the expression of p27Kip1, which inhibits CDKs regulating each phase of the cell cycle. Importantly, we also determined that elevating SOX2 inhibits the growth of MB, PCa, and PDAC xenografts. Significantly, when Dox was withdrawn and SOX2 returned to endogenous levels, tumor growth rapidly resumed and paralleled the growth kinetics of control tumors. Conclusion: Our findings demonstrate that elevating SOX2 significantly inhibits the growth of a diverse group of tumor types. Significantly, SOX2, when elevated, appears to function as a master regulator of the cell cycle machinery by downregulating a broad spectrum of cyclins and CDKs. Additionally, our inducible model of tumor growth arrest provides a novel platform to investigate the biology of quiescent tumor cells and the testing of clinically used drugs to target quiescent/dormant tumor cells in vivo. Citation Format: Ethan P. Metz, Erin L. Wuebben, Phillip J. Wilder, Angie Rizzino. Sox2 elevation downregulates a broad spectrum of cell cycle machinery and induces a reversible state of tumor growth arrest [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5815.

Corrigendum.

Development, Growth & DifferentiationVolume 62, Issue 6 p. 462-462 CORRIGENDUMFree Access Corrigendum This article corrects the following: Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming Asuka Takehara, Yasuhisa Matsui, Volume 61Issue 6Development, Growth & Differentiation pages: 357-364 First Published online: June 14, 2019 First published: 26 August 2020 https://doi.org/10.1111/dgd.12672AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Takehara and Mtasui (2019), the authors note that, in the abstract, a sentence ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally repressed by p53, was significantly downregulated by AKT activation.’ should be ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation.’ The authors would like to apologize for any inconvenience caused. REFERENCE Takehara, A, & Mtasui, Y (2019). Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming. Development Growth and Differentiation, 61(6), 357– 364. https://doi.org/10.1111/dgd.12621 Volume62, Issue6August 2020Pages 462-462 ReferencesRelatedInformation

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity.

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin-dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild-type (WT) mice: exhibiting higher viral load in lung tissue, faster body-weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN-β and several critical antiviral interferon-stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV-infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN-β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.

PTEN Inhibition Accelerates Corneal Endothelial Wound Healing through Increased Endothelial Cell Division and Migration.

PurposeTo investigate the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of corneal endothelial cell (CECs) focusing on proliferation and migration, and to further evaluate the application of PTEN inhibitors in the treatment of corneal endothelial dysfunction in a rat model.MethodsExpression of PTEN in human and rat corneal endothelium was determined by immunocytochemistry, western blotting, and ELISA. A small molecular inhibitor of PTEN, bpV(pic), was applied in the culture of human CEC cell line B4G12 and organ-cultured rat cornea in the presence of transforming growth factor beta 2 (TGF-β2). Cell cycle status was detected by flow cytometry and BrdU staining. Subcellular localization for endogenous p27Kip1 was detected by immunocytochemistry and western blotting. Moreover, exogenous transfected YFP-p27Kip1 was observed under a fluorescent microscope. Cell migration was examined with a wound scratch model and transwell invasion assay. Finally, bpV(pic) was intracamerally injected in a rat corneal endothelial injury model. The wound healing process was evaluated by slit lamp biomicroscopy, optical coherence tomography, histological and scanning electron microscope examination.ResultsThe expression of PTEN in human corneal endothelium was higher compared with rat, which we speculate was mostly responsible for the relatively less proliferation capacity of human CEC than rat. PTEN inhibition by bpV(pic) could reverse TGF-β2-induced CEC G1-arrest by alleviating p27Kip1 nuclear accumulation and decreasing total p27Kip1 expression. In addition, bpV(pic) promoted CEC migration, which acted synergistically with TGF-β2. Finally, intracameral injection of bpV(pic) could promote corneal endothelial wound healing in a rat model.ConclusionsOur study provided experimental basis for the development of therapeutic agent targeting on PTEN for the treatment of corneal endothelial dysfunction.

Abstract A74: The HIV protease inhibitor nelfinavir, alone or in combination with the proteasome inhibitor bortezomib, is cytotoxic to high-grade serous ovarian cancer cells regardless of platinum sensitivity

Abstract High-grade serous ovarian cancer (HGSOC) is a deadly disease, affecting women worldwide. Major challenges in treating HGSOC patients involve overcoming resistance to platinum (Pt)-based therapy by developing novel treatment approaches. Nelfinavir (NFV), an anti-HIV drug, has shown to be effective against cancers of high secretory capacity—e.g., multiple myeloma—by aggravating the stress of the endoplasmic reticulum (ER). Its potential therapeutic role against HGSOC, however, remains unclear. We hypothesized that simultaneous ER stress aggravation by NFV and blockage of the proteolytic capacity of the proteasome with a proteasome inhibitor should cause sufficient ER stress to tilt cells to a death fate. In this study, we investigated the cytotoxicity of the ER aggravator NFV as monotherapy and in combination with the proteasome inhibitor bortezomib (BZ) against HGSOC cells having differential sensitivities to cisplatin (CDDP). NFV caused cell cycle arrest associated with increased expression of cyclin-dependent kinase inhibitor p27kip1, while triggering the unfolded protein response (UPR) in a dose-dependent manner and assessed by increased subrogate ER stress biomarkers GRP78 and CHOP. We also discovered that blocking the ubiquitin proteasome system (UPS) using cytostatic concentrations of BZ causes accumulation of poly-ubiquitinated proteins and further activation of the UPR. More importantly, when we combined BZ with NFV, we observed a potentiation of the action of BZ leading to HGSOC lethality regardless of Pt sensitivity. These results suggest that NFV could potentially be used following standard Pt-based treatment, either as monotherapy or in conjunction with proteasome inhibitors, to kill the remaining HGSOC cells that otherwise recur and recreate the disease. In addition, the data provide the rationale for rapid repurposing to treat HGSOC of two drugs clinically approved for other uses, as their systemic toxicities have already been assessed. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Michael A. Lisio, Carlos M. Telleria. The HIV protease inhibitor nelfinavir, alone or in combination with the proteasome inhibitor bortezomib, is cytotoxic to high-grade serous ovarian cancer cells regardless of platinum sensitivity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A74.

AMPKα2 deficiency exacerbates hypoxia-induced pulmonary hypertension by promoting pulmonary arterial smooth muscle cell proliferation.

Increased evidence indicates that adenosine monophosphate-activated protein kinase (AMPK) plays a vital role in vascular homeostasis, especially under hypoxia, and protects against the progression of pulmonary hypertension (PH). However, the role of AMPK in the pathogenesis of PH remains to be clarified. In the present study, we confirmed that a loss of AMPKα2 exacerbated the development of PH by using hypoxia-induced PH model in AMPKα2 -/- mice. After a 4-week period of hypoxic exposure, AMPKα2 -/- mice exhibited more severe pulmonary vascular remodeling and pulmonary vascular smooth muscle cell (SMC) proliferation when compared with wild type (WT) mice. In vitro, AMPKα2 knockdown promoted the proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. This phenomenon was accompanied by upregulated Skp2 and downregulated p27kip1 expression and was abolished by rapamycin, an inhibitor of mTOR. These results indicate that AMPKα2 deficiency exacerbates hypoxia-induced PH by promoting PASMC proliferation via the mTOR/Skp2/p27kip1 signaling axis. Therefore, enhanced AMPKα2 activity might underlie a novel therapeutic strategy for the management of PH.

Role of pyroglutamylated RFamide peptide43 in germ cell dynamics in mice testes in relation to energy metabolism

QRFP is a neuropeptide that regulates glucose homeostasis and increases insulin sensitivity in tissues. We have previously shown that QRFP and its receptor (GPR103) are predominantly expressed in germ cells and Sertoli cells, respectively, in mice testes. In the present study, we report that QRFP caused an increase in PCNA and a decrease in p27Kip1 expressions in the testis under both in vivo and ex vivo conditions. Besides, via an in vivo study, cell cycle analysis by FACS showed an increase in 2C cells and a decrease in 1C cells. QRFP also induced expression of GDNF and phosphorylation of Akt and ERK-1/2. Together these results suggest that QRFP has a proliferative effect on germ cells in mice testes, since it caused a proportional increase in the mitotic activity and the number of spermatogonial cells. Further, observations of increased expressions of STAT-3 and Neurog3 in treated mice suggest that QRFP treatment regulates priming of undifferentiated spermatogonia to undergo differentiation, while a decrease in c-Kit expression indicate that spermatogonia at this time point are in an undifferentiated state. In addition, QRFP administration also caused an increase in intratesticular levels of glucose and lactate, and in LDH activity accompanied by increased expressions of GLUT-3 and LDH-C in the testis. Also, the phosphorylation of IR-β and expressions of p-Akt and p-mTOR were increased under ex vivo conditions in testicular tissue. In conclusion, our findings suggest that QRFP treatment caused proliferation of germ cells independently from the hypothalamic-pituitary axis via regulation of testicular energy metabolism.

Clinical significance of p27 Kip1 expression in advanced ovarian cancer

BackgroundOvarian cancer is the most common gynecological malignancy. In patients with advanced ovarian cancer, some biological parameters have prognostic implementations. P27kip1 is an inhibitor of a cycline-dependent kinase, its loss, can contribute to tumor progression.ObjectiveThis study aimed to examine the importance of P27KIP1 protein in predicting the prognosis and response to neoadjuvant chemotherapy in patients with advanced ovarian epithelial cancer and to compare the outcomes of immunohistochemistry with Quantitative Real-time PCR.Patients and methodsWe have studied P27KIP1expression by both immunohistochemistry and Quantitative Real-time PCR from 88 patients with advanced ovarian carcinomas undergone radical debulking surgery and received Paclitaxel followed by Cisplatin every 3 weeks for a total of 6 cycles. We also studied their association with both chemotherapy response and patient survival.ResultsNuclear expression of p27KIP1 protein was intense in 86 normal ovarian tissues and 42 of 88 carcinomas. The P27kip1mRNA expression level by qRT-PCR was very low in ovarian cancer tissues relative to its adjacent normal tissues. The results were statistically significant by both methods of determination. p27KIP1 expression was significantly related to good prognostic parameters as low stage tumors, differentiated tumors, absence of ascites, residual disease < 2 cm, and response to chemotherapy but not with histopathological type in case of determination by immunohistochemistry. Comparison of P27kip1 by both immunohistochemistry and qRT-PCR with different prognostic parameters revealed no significant difference between both methods in the assessment of these parameters. In 4 years of follow-up, 20.5% of patients were alive without evidence of disease. 6.8% were alive with disease. The disease-related four -year survival rate for the whole group was 28.2%. In multivariate analysis, residual disease, histological type, tumor differentiation, ascites was of independent prognostic significance.ConclusionIn ovarian cancer, patients with loss of p27KIP1 expression are at a greater likelihood of disease progression, p27KIP1 may be used as a molecular marker to predict response to chemotherapy and prognosis. Both immunohistochemistry and qRT-PCR have equal reliability in the determination of p27 KIP1.

A novel CDK-independent function of p27Kip1 in preciliary vesicle trafficking during ciliogenesis

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86–140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.

Overexpression of negative regulator of ubiquitin-like proteins 1 (NUB1) inhibits proliferation and invasion of gastric cancer cells through upregulation of p27Kip1 and inhibition of epithelial-mesenchymal transition

BackgroundGastric cancer (GC), one of the most common causes of malignant tumors, is characterized by a high degree of heterogeneity, which represents a bottleneck in gaining comprehensive insights into its pathogenesis. Negative regulator of ubiquitin-like proteins 1 (NUB1) is a transcription factor that negatively regulates ubiquitylation system. Although the abnormal expression of NUB1 has been reported in many types of cancer, its expression pattern and functions in GC are poorly understood. Materials and methodsThe link between NUB1 expression and clinicopathological characteristics was analyzed by immunohistochemical staining, and the suitability of NUB1 as a prognostic marker was explored using a public database on mRNA expression levels. NUB1 overexpression was performed by lentiviral transfection. Cell proliferation was estimated using the cell counting kit-8 (CCK-8) assay. The effect on NUB1 on cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Real-time PCR (RT-PCR) and western blotting experiments were used to explore the mechanism of p27Kip1 regulation by NUB1. Cell migration and invasion were determined by wound healing and transwell assays, respectively. Expression levels of epithelial-mesenchymal transition (EMT) indicator proteins were determined by western blotting. ResultsIn this study, based on a comparative analysis of cancer tissues from 116 post-operative GC patients with the respective paracancerous healthy tissues, we found that NUB1 was downregulated in GC tissues. At the same time, a low expression level of NUB1 was closely related to poor prognosis. Results from In vitro cancer cell experiments verified that overexpressed NUB1 inhibited GC proliferation, migration, and invasion. In addition, NUB1 upregulated the expression of p27Kip1 and blocked the G1/S phase transition in cell cycle. Finally, NUB1 inhibited the process of EMT by upregulating E-cadherin and downregulating N-cadherin, vimentin, and matrix metalloproteinase-2 (MMP-2). ConclusionReduced NUB1 levels were positively associated to poor prognosis of GC, whereas NUB1 overexpression inhibited the proliferation and blocked the G1/S phase transition in GC cells. This may be strongly coupled to the post-translational modification mechanism (PTM), which could, in turn, reduce the level of ubiquitinylated p27Kip1 and upregulate its expression. In addition, NUB1 overexpression inhibited GC migration and invasion by regulating EMT. In view of the positive tumor-suppressive effect of NUB1 on GC occurrence and progression reported here, this study enhances our understanding of the molecular mechanism of NUB1-mediated GC regulation, and may provide insights into novel drug targets or anti-tumor strategies with better accuracy and efficacy.

Hippocampal Neurogenesis Requires Cell-Autonomous Thyroid Hormone Signaling.

SummaryAdult hippocampal neurogenesis is strongly dependent on thyroid hormone (TH). Whether TH signaling regulates this process in a cell-autonomous or non-autonomous manner remains unknown. To answer this question, we used global and conditional knockouts of the TH transporter monocarboxylate transporter 8 (MCT8), having first used FACS and immunohistochemistry to demonstrate that MCT8 is the only TH transporter expressed on neuroblasts and adult slice cultures to confirm a necessary role for MCT8 in neurogenesis. Both mice with a global deletion or an adult neural stem cell-specific deletion of MCT8 showed decreased expression of the cell-cycle inhibitor P27KIP1, reduced differentiation of neuroblasts, and impaired generation of new granule cell neurons, with global knockout mice also showing enhanced neuroblast proliferation. Together, our results reveal a cell-autonomous role for TH signaling in adult hippocampal neurogenesis alongside non-cell-autonomous effects on cell proliferation earlier in the lineage.

Souchinone Inhibits Mesangial Cell Fibrosis and Inflammatory Response Through TGF‐β1/Smad signaling and NLRP3 inflammasome pathway

Mesangial proliferative glomerulonephritis is characterized by an increased number of mesangial cells in the glomeruli in the kidneys and damage to the glomeruli by activation of some growth factors (e.g., angiotensin II) and deposition of extracellular matrix proteins. However, the effect of sauchinone on mesangial proliferative glomerulonephritis is still unclear. In the present study, we investigated the protective effect of sauchinone against angiotensin II (AngII)‐induced proliferation and fibrosis in human renal mesangial cells. Thus, this study was investigated the inhibitory effect of sauchinone (0.1–1 μM) on AngII‐stimulated mesangial cells proliferation and fibrosis. Sauchinone, one of the active lignan isolated from Saururus chinensis, was reported to possess broad bioactivities, such as anti‐inflammatory, anti‐oxidant, anti‐tumor, anti‐apoptosis and anti‐obesity effects.In this study, thymidine incorporation under AngII was significantly accelerated, which was inhibited by sauchinone in a dose dependent manner. In migration assays, a significant reduction in wound surface was observed in pretreatment of sauchinone. Sauchinone induced down‐regulation of cyclins/CDKs and up‐regulation of CDK inhibitor, p21waf1/cip1 and p27kip1 expression. In addition, AngII enhanced expression of fibrosis biomarkers such as collagen IV and CTGF, which was markedly attenuated by sauchinone. Sauchinone decreased TGF‐β1 and Smad‐2/Smad‐4 expression, whereas increased Smad‐7 expression under AngII. Furthermore, sauchinone inhibited AngII‐induced inflammatory factors level as well as NF‐κB p65 translocation and ROS production.These results suggested that sauchinone may alleviate mesangial proliferation and inflammation possibly involved in renal fibrotic process, further mesangial proliferative glomerulonephritis through disturbing TGF‐β1/Smad signaling and NLRP3 inflammasome pathway. Thus, sauchinone might prove to be effective in the treatment of renal dysfunction leading to mesangial proliferative glomerulonephritis.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A5A2015805)(2018R1D1A1B07050720).