P21 Promoter Research Articles

Abstract 962: Small-molecule NSC58874 releases and activates p73 via induction of mutant p53 degradation in cancer cells

Abstract The tumor suppressor p53 is the most frequently mutated gene in cancer. Mutant p53 is a challenge to target in cancer therapy due to its many mutations and the fact that it is an inactivated transcription factor. p73 is a member of the p53 family but very rarely mutated in cancer cells. Therefore, activation of p73 is one of the promising strategies to restore the p53 pathway signaling in cancer therapy. Our laboratory has reported that p73 can be activated by small molecules to restore p53 pathway signaling via induction of p73 expression or interruption of p73 interaction with mutant p53. In the present study, we explored a strategy for releasing p73 from a mutant p53 inhibitory complex by induction of mutant p53 degradation using small molecule NSC59984. We previously reported that NSC59984 induces mutant p53 degradation and restores p53 pathway signaling through p73 in p53-mutant cancer cells. To address the mechanism of whether p73 is released from a mutant p53 inhibitory complex to transcriptionally regulate p53 targets, we performed p53-RE-luc reporter assays and Chromatin Immunoprecipitation (ChIP)-PCR in cancer cells with overexpression of p73. We found NSC59984 enhances p73-mediated signaling based on p53 target (such as p21 and noxa) induction at the protein level and p53-RE-luc reporter assay. Further ChIP-PCR analysis showed that NSC59984 treatment increases p73-binding to the p21 and Noxa promoters. These results taken together suggest that NSC59984 enhances p73 transcriptional activity to restore the p53 pathway signaling. It is also well known that p73 activity is regulated through a complex mechanism such as post-translational modifications and protein-protein interactions. We found that NSC59984 can stimulate the ERK2 signaling pathway. The MEK1 inhibitor U0126 partially blocked p73 binding to the p21 and Noxa promoters and their gene expression in cells treated with NSC59984, and this correlated with the rescue of mutant p53 stabilization. These results suggest that p73 is released from mutant p53 inhibitory complex and further stimulated through ERK2 signaling, as a mechanism of tumor suppression in tumors with mutant p53. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Small-molecule NSC58874 releases and activates p73 via induction of mutant p53 degradation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 962.

Abstract 2489: A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis

Abstract Two of the most common events in human tumors are mutation of the tumor suppressor gene TP53 and development of aneuploidy. In addition to losing their wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mechanistic understanding of mutant p53 GOF activities is complicated by the diversity and context-specific nature of reported GOF phenotypes. The study of mutant p53 GOF activities is especially challenging because mutations in p53 are positively correlated with the development of aneuploidy, which can increase heterogeneity through diverse chromosomal alterations and itself contributes to tumorigenesis. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic epithelial cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Additionally, the use of clonally-derived cell lines originating from the same near diploid parental genetic background allows for assessment of the genomic alterations and resulting molecular heterogeneity following mutation of TP53. Through genomic, transcriptomic, and cellular based assays we have validated our cell line models and found that missense mutant and p53 null cells display loss of p53 function. Through functional genomics analyses comparing isogenic epithelial cells, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and aneuploidy and assessed whether our clonal cell lines display several previously reported mutant p53 GOF phenotypes such as altered gene expression, proliferation, metabolism, drug sensitivity, and migration. Further, using lentiviral mediated knockdown of p53 protein we evaluated the dependency of these phenotypes on expression of mutant p53 protein. Finally, data from The Cancer Genome Atlas (TCGA) was used for the analysis of manifestations of clinical mutant p53 GOF phenotypes. The dissection of mutant p53 GOF phenotypes will improve the current understanding of the role of mutant p53 in tumorigenesis. The results generated from these studies have the potential for clinical translation in major types of human cancer that have high-frequency p53 mutation. Citation Format: Lindsay N. Redman-Rivera, Timothy M. Shaver, Hailing Jin, Johanna M. Schafer, Quanhu Sheng, Rachel A. Hongo, Kathryn E. Beckermann, Brian D. Lehmann, Ferrin C. Wheeler, Jennifer A. Pietenpol. A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2489.

Prostaglandin E2 Protects HSC from DNA Damage Induced By Ionizing Radiation By Preventing Generation of Reactive Oxygen Species and Histone Acetylation

The hematopoietic system is highly radiosensitive; thus therapeutic and unwanted irradiation can produce significant/lethal acute and delayed bone marrow injury. In a murine hematopoietic-acute radiation syndrome (H-ARS) model, a single dose of 16,16 dimethyl Prostaglandin E2 (dmPGE2) given 15 min to 3 hrs before lethal (LD50/30-90/30) total body irradiation (TBI) results in 100% survival of mice at 30 days, with accelerated recovery of WBC, and hematopoietic stem and progenitor cell number and function.To explore mechanisms responsible for the radio-protective effect(s) of dmPGE2 we evaluated hematopoietic cells at early points post-TBI, focusing on oxidative pathways and epigenetic changes. Hematopoietic cells were isolated and evaluated at 1 hr and 1, 3 and 9 days post LD50 [853 cGy] TBI. Mitochondrial ROS and membrane potential were measured by flow cytometry in SLAM-SKL cells. Protein levels of gH2AX, LC3b, BNIP3, P16, P53, P62 acetylated H3K9 and H4K16 histones and SIRT1 were measured by flow cytometry. Acetylation of H3K9 and H4K16 at the KU70, Ku80, LC3b, Atg5, Atg7, Atg12, BNIP3, NF-kB and p53 promoters was evaluated by ChIP.Lethal radiation significantly increased HSC mitochondrial ROS and reduced membrane potential at 1,3 and 9 days post-TBI. Significant DNA damage was observed at 1 hour as measured by gH2AX. In contrast, mitochondrial ROS and DNA damage (gH2AX) were significantly lower in mice that received dmPGE2 before irradiation. In vehicle treated irradiated mice, we observed hyper-acetylated H3K9 and H4K16 at the Ku70/80 promoter, suggesting diminished capacity of Ku70/80 to repair DNA damage. In contrast, in mice treated with dmPGE2, H3K9 and H4K16 acetylation was significantly lower. Reduced mitochondrial membrane potential seen in vehicle treated mice was also prevented. The senescence markers SA-beta-gal and P16 in HSC were significantly reduced in PGE2 treated mice.The sirtuin family NAD-dependent deacetylase, SIRT1, has broad functions including gene silencing and DNA repair. In control irradiated mice SIRT1 levels dramatically decreased over 9 days post-irradiation, but remained significantly higher in dmPGE treated mice, which may account for the low acetylation levels of H3K9 and H4K16 in PGE treated mice. Consistent with elevated SIRT1 in PGE2 treated mice, we also observed global deacetylation of H3K9 and H4K16 in lineage negative and SLAM LSK cells at 1-3 days post-TBI. P53 is a key transcriptional regulator of genes involved in cell cycle, apoptosis and DNA repair and is acetylated after DNA damage. In control mice, irradiation resulted in robust acetylation of histone proteins at the p53 promoter, whereas dmPGE2 administration prevented acetylation of H4K16, but not H3K9. In addition, dmPGE2 blocked p53-mediated apoptosis via repression of p53 transcription.Since SIRT1 promotes autophagy, we evaluated autophagy related genes. CHIP analyses revealed low levels of acetylated-H3K9 at the LC3B, ATG7 and NF-KB promoters and deacetylated H4K16 at the LC3B, ATG7, BNIP3 and NF-kB promoters in PGE treated mice compared to vehicle controls. No effects on histone deacetylation of the Atg5 and Atg12 genes. BNIP3 is a mitophagy marker that when elevated downregulates mitochondrial mass and respiration. In irradiated control mice, BNIP3 levels were decreased at 1through 9 days, but remained elevated in mice treated with dmPGE2. The autophagy substrate P62 was significantly increased at 1 and 3 days post-irradiation in control mice, but was significantly lower in PGE2 treated mice. SIRT1 activation also leads to decreased binding of NFkB-p65 to DNA and attenuated oxidative stress through reduced transcription. CHIP data showed that TBI increased acetylation of H3K9 and H4K16 at the NF-kB p65 promoter in vehicle treated mice, whereas hyperacetylation of H3K9 and H4K16 and NF-kB transcription did not occur in mice treated with dmPGE2 before irradiation.These studies suggest that the radio protective effect of dmPGE2 and accelerated recovery of hematopoiesis following radiation exposure is mediated in part through reduction in oxidative stress and DNA damage induced in HSC and epigenetically, through prevention of histone acetylation of genes associated with DNA repair, autophagy and mitophagy, likely mediated through elevation of SIRT1. DisclosuresNo relevant conflicts of interest to declare.

The Impacts of Transcriptional Regulatory Mechanism and Protein Interaction Relationship between RUNX1 and KLF4 on the Biological Functions of t (8;21) Leukemia Cells

Our previous study has reported that sodium phenylbutyrate (PB), one of the HDAC inhibitors, could induce t (8;21) leukemia cells to undergo differentiation and apoptosis. However, genes that promote differentiation and apoptosis in this process are not fully identified. Identification of these genes and elucidation of their interaction relationships are essential to develop novel therapy strategies for t (8;21) leukemia.In this study, we found that the expressions of RUNX1, KLF4 and P57 at both mRNA and protein level were significantly up-regulated in Kasumi-1 cells after PB treatment. Bioinformatics analysis further demonstrated that there might be some transcriptional regulatory mechanism and protein interaction relationship among these three genes.For the study of transcriptional regulatory mechanism, a KLF4 promoter/enhancer reporter plasmid containing RUNX1 binding sites and a P57 promoter/enhancer reporter plasmid containing KLF4 binding sites were constructed. Luciferase assay showed that RUNX1 exhibited strong transcriptional activation effect on KLF4 promoter/enhancer reporter plasmid in a dose-dependent manner, while RUNX1-ETO showed slight effect. KLF4 exhibited dose-dependent transcriptional activation effect on P57 promoter /enhancer reporter plasmid. These transcriptional regulatory relationships were further confirmed by Western blot assay. Transient expression of exogenous RUNX1 up-regulated the expression of KLF4 and P57 in 293T cells and Kasumi-1 cells, while RUNX1-ETO had no effect.In the study of protein interaction relationship, co-immunoprecipitation (CO-IP) and immunofluorescence confocal imaging showed that RUNX1 interacted with KLF4 and co-localized with it in nucleus, and Runt homology domain (RHD) was responsible for this interaction. RUNX1-ETO competed with RUNX1 for binding to KLF4 in a dose-dependent manner through RHD and the ETO domains. PB has no effect on the interactions between RUNX1/RUNX1-ETO with KLF4. To further investigate the effects of RUNX1 and RUNX1-ETO on KLF4-dependent transactivation of target genes, a luciferase reporting plasmid containing KLF4 binding sites was constructed. Luciferase assay was then performed and showed that RUNX1 could coactivate KLF4 target genes while RUNX1-ETO had little effect.Finally,RUNX1, KLF4 and P57 were overexpressed in Kasumi-1 cells by a pCDH lentivirus system, and their biological functions were investigated by MTS assay and flow cytometry. Overexpression of either of the three genes could inhibit cell proliferation, block cell cycle and induce apoptosis of t (8;21) leukemia cells. Moreover, in vivo experiment demonstrated that overexpression of KLF4 and P57 in mouse bone marrow leukemic cells could reduce the incidence of leukemia development in recipient mice.In conclusion, we reported “RUNX1-KLF4-P57” as a novel signaling pathway in t (8;21) leukemia cells in this study. RUNX1 transactivated KLF4 and then P57 in leukemia cells to inhibit cell proliferation, block cell cycle and induce cell apoptosis, while oncoprotein RUNX1-ETO had little effect. Moreover, RUNX1 interacted with KLF4 and coactivated its target genes, which could be blocked by the competed binding of RUNX1-ETO with KLF4. Our study revealed the regulatory relationship between RUNX1 and KLF4 and the impacts of RUNX1-ETO on this relationship, which helps to elucidate the mechanism of t (8;21) leukemogenensis. DisclosuresNo relevant conflicts of interest to declare.

LncRNA LINC00525 suppresses p21 expression via mRNA decay and triplex-mediated changes in chromatin structure in lung adenocarcinoma.

BackgroundEmerging evidence suggests that long noncoding RNAs (lncRNAs) play crucial roles in various cancers. In the present study, we aim to investigate the function and molecular mechanism of an up‐regulated and survival‐associated lncRNA, LINC00525, in lung adenocarcinoma (LUAD).MethodsThe expression level of LINC00525 in tissues was determined by quantitative reverse transcription polymerase chain reaction (RT‐qPCR) and in situ hybridization (ISH). The functional role of LINC00525 in LUAD was investigated using gain‐and loss‐of‐function approaches, both in vivo and in vitro. RNA pull‐down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), triplex‐capture assay, dual‐luciferase assay, gene expression microarray, and bioinformatics analysis were used to investigate the potential underlying mechanisms involved.ResultsLINC00525 is highly expressed in LUAD cells and tissues. Survival analysis indicated that upregulation of LINC00525 was associated with poor prognosis in patients with LUAD patients. Knockdown of LINC00525 inhibited cell proliferation and cell cycle progression in vitro. In xenograft models, LINC00525 knockdown suppressed tumor growth and tumorigenesis of tumor‐bearing mice. Mechanistically, LINC00525 epigenetically suppressed p21 transcription by guiding Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) to the p21 promoter through an formation of RNA‐DNA triplex with the p21 promoter, leading to increased trimethylation of lysine 27 on histone 3 (H3K27me3) of the p21 promoter. In addition, LINC00525 repressed p21 expression post‐transcriptionally by enhancing p21 mRNA decay. LINC00525 promoted p21 mRNA decay by competitively binding to RNA Binding Motif Single Stranded Interacting Protein 2 (RBMS2).ConclusionOur findings demonstrate that LINC00525 promotes the progression of LUAD by reducing the transcription and stability of p21 mRNA in concert with EZH2 and RBMS2, thus suggesting that LINC00525 may be a potential therapeutic target for clinical intervention in LUAD.

TNFα Enhances Tamoxifen Sensitivity through Dissociation of ERα-p53-NCOR1 Complexes in ERα-Positive Breast Cancer.

Simple SummaryTamoxifen has been clinically applied as a central chemotherapeutic agent for treatment of estrogen receptor (ER)-positive breast cancer. However, many ER-positive breast cancer patients with the high ER level demonstrate intrinsic resistance against the tamoxifen therapy. The aim of our study was to find an effective approach to enhance tamoxifen sensitivity. We found that tumor necrosis factor α (TNFα) has a potential to overcome tamoxifen resistance through disruption of nuclear receptor corepressor 1 (NCOR1)-p53-ERα complexes in ER-positive MCF7 xenograft mice. NCOR1 knock-down with TNFα treatment induced ERα destabilization and increased the occupancy of p53 at the p21 promoter. Finally, we confirmed the combinational application with tamoxifen, TNFα and short-hairpin NCOR1 showed the enhanced suppressive effect of tumor growth in MCF xenograft mice compared to single tamoxifen treatment. These results provide a possibility for application of NCOR1 as a putative therapeutic target to overcome tamoxifen resistance in ERα-positive breast cancer.Tamoxifen is widely used as a medication for estrogen receptor α (ERα)-positive breast cancer, despite the ~50% incidence of tamoxifen resistance. To overcome such resistance, combining tamoxifen with other agents is considered an effective approach. Here, through in vitro studies with ER-positive MCF7 cells and ER-negative MDA-MB-231 cells, validated by the use of xenograft mice, we investigated the potential of tumor necrosis factor α (TNFα) to enhance tamoxifen sensitivity and identified NCOR1 as a key downstream regulator. TNFα specifically degraded nuclear receptor corepressor 1 (NCOR1) in MCF7 cells. Moreover, knockdown of NCOR1, similar to TNFα treatment, suppressed cancer cell growth and promoted apoptosis only in MCF7 cells and MCF7 xenograft mice through the stabilization of p53, a tumor suppressor protein. Interestingly, NCOR1 knockdown with TNFα treatment increased the occupancy of p53 at the p21 promoter, while decreasing that of ERα. Notably, NCOR1 formed a complex with p53 and ERα, which was disrupted by TNFα. Finally, combinatorial treatment with tamoxifen, TNFα and short–hairpin (sh)-NCOR1 resulted in enhanced suppression of tumor growth in MCF7 xenograft mice compared to single tamoxifen treatment. In conclusion, TNFα promoted tamoxifen sensitivity through the dissociation of the ERα-p53-NCOR1 complex, pointing at NCOR1 as a putative therapeutic target for overcoming tamoxifen resistance in ERα-positive breast cancer.

Capsanthin induces G1/S phase arrest, erlotinib-sensitivity and inhibits tumor progression by suppressing EZH2-mediated epigenetically silencing of p21 in triple-negative breast cancer cells.

Capsanthin is a naturally occurring red pepper carotenoid with possible antitumor activity, but its antitumor mechanisms have yet to be delineated. We tested the anti-proliferative activity of capsanthin with human triple-negative breast cancer (TNBC) and found that cell proliferation was inhibited after 24, 48 and 72 h of treatment. We also investigated the cellular and molecular mechanisms of the antitumor efficacy of capsanthin on TNBC cells and found that capsanthin delayed cell-cycle progression at the G1/S stage, that cyclin A expression was suppressed, and that p21 expression was upregulated. Capsanthin also inhibited the EZH2 expression and EZH2 could binding to the p21 promoter in TNBC cells. We further discovered that capsanthin has synthetic effects when combined with erlotinib (Tarceva). In the animal experiment, we found that the capsanthin-induced inhibition of TNBC cell proliferation decreased the incidence of the initiation and growth of TNBC cell–derived tumors in mice. Our study reveals that capsanthin exerted antitumor effects through delaying cell-cycle progression, induces erlotinib-sensitivity and inhibits tumor progression by inhibiting EZH2/p21 axis, and capsanthin is a potential drug candidate for development of a safe and effective therapy against TNBCs, especially for TNBCs that have developed resistance to targeting therapy.

GIPC2 is an endocrine-specific tumor suppressor gene for both sporadic and hereditary tumors of RET- and SDHB-, but not VHL-associated clusters of pheochromocytoma/paraganglioma

Pheochromocytoma/paraganglioma (PPGL) is an endocrine tumor of the chromaffin cells in the adrenal medulla or the paraganglia. Currently, about 70% of PPGLs can be explained by germline or somatic mutations in several broadly expressed susceptibility genes including RET, VHL, and SDHB, while for the remaining, mainly sporadic cases, the pathogenesis is still unclear. Even for known susceptible genes, how mutations in these mostly ubiquitous genes result in tissue-specific pathogenesis remains unanswered, and why RET-mutated tumors almost always occur in the adrenal while SDHB-mutated tumors mostly occur extra-adrenal remains a mystery. By analyzing 22 sporadic PPGLs using SNP 6.0 genotyping arrays combined with expression profiling of 4 normal and 4 tumor tissues, we identified GIPC2, a gene located at 1p31.1 with preferential expression in adrenal and inducible by adrenal glucocorticoid, as a novel putative tumor suppressor gene for PPGLs. Copy number deletion and GIPC2 promoter hypermethylation but not GIPC2 mutation, accompanied with reduced GIPC2 expression, were observed in 39 of 55 PPGLs in our cohort. Examination of a published expression database consisting of 188 PPGLs found little GIPC2 expression in Cluster 1A (SDHx-associated) and Cluster 2A (NF1/RET-associated) tumors, but less pronounced reduction of GIPC2 expression in Cluster 1B (VHL-associated) and Cluster 2B/2C tumors. GIPC2 induced p27, suppressed MAPK/ERK and HIF-1ɑ pathways as well as cancer cell proliferation. Overexpressing GIPC2 in PC12 cells inhibited tumor growth in nude mice. We found GIPC2 interacted with the nucleoprotein NONO and both proteins regulated p27 transcription through the same GGCC box on p27 promoter. Significantly, low expression of both GIPC2 and p27 was associated with shorter disease-free survival time of PPGLs patients in the TCGA database. We found that PPGL-causing mutations in RET and in SDHB could lead to primary rat adrenal chromaffin cell proliferation, ERK activation, and p27 downregulation, all requiring downregulating GIPC2. Notably, the RET-mutant effect required the presence of dexamethasone while the SDHB-mutant effect required its absence, providing a plausible explanation for the tumor location preference. In contrast, the PPGL-predisposing VHL mutations had no effect on proliferation and GIPC2 expression but caused p53 downregulation and reduced apoptosis in chromaffin cells compared with wild-type VHL. Thus, our study raises the importance of cortical hormone in PPGL development, and GIPC2 as a novel tumor suppressor provides a unified molecular mechanism for the tumorigenesis of both sporadic and hereditary tumors of Clusters 1A and 2A concerning SDHB and RET, but not tumors of Cluster 1B concerning VHL and other clusters.

The role of E26 transformation-specific variant transcription factor 5 in colorectal cancer cell proliferation and cell cycle progression

E26 transformation-specific variant transcription factor 5 (ETV5) contributes to tumor growth and progression and promotes colorectal cancer (CRC) angiogenesis. Previous studies indicate that ETV5 may regulate the cell cycle, but its detailed mechanism remain unclear. Gene Ontology (GO) analysis of RNA-seq data revealed that ETV5 possibly regulates the cell cycle in CRC. Here, in vitro and in vivo experiments were performed to verify that ETV5 promoted tumor progression and influenced cell cycle G1/S transition. Cell cycle PCR array and co-immunoprecipitation (Co-IP) helped identify the p21-CDKs pathway. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to determine whether ETV5 binds to the p21 promoter. ETV5 and p21 were detected by immunohistochemistry, and the effects of their expression on CRC patients were evaluated. ETV5 upregulation enhanced tumor proliferative capacity and promoted G1 phase transfer to the S phase. ETV5 knockdown slowed the growth of CRC cells and repressed the G1/S transition. We also found p21 as a downstream target of ETV5. p21 knockdown resulted in faster CRC cell growth and in more cells being driven from the G0/1 phase into the S phase. Co-IP experiments showed that p21 banding to CDK2, CDK4, and CDK6 inhibited p130 phosphorylation. Using the ChIP and luciferase reporter assay, we confirmed that ETV5 bound to the p21 promoter and repressed p21 expression. CRC patients with high ETV5 expression and low p21 expression showed the worst prognosis. Finally, by targeting p21 to regulate CDK function, ETV5 also changed drug-sensitivity to palbociclib and dinaciclib. In conclusion, ETV5 promoted cell cycle G1/S transition through transcriptional inhibition of p21, thereby accelerating tumor growth. Moreover, ETV5 changed drug-sensitivity to palbociclib and dinaciclib. Therefore, therapeutic regimens targeting ETV5 may be promising in improving the efficacy of target-CDK treatment in CRC.

LMNB2 promotes the progression of colorectal cancer by silencing p21 expression

Colorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

MiR-181d/RBP2/NF-κB p65 Feedback Regulation Promotes Chronic Myeloid Leukemia Blast Crisis

BackgroundChronic myeloid leukemia (CML) is a malignant clonal proliferative disease. Once it progresses into the phase of blast crisis (CML-BP), the curative effect is poor, and the fatality rate is extremely high. Therefore, it is urgent to explore the molecular mechanisms of blast crisis and identify new therapeutic targets.MethodsThe expression levels of miR-181d, RBP2 and NF-κB p65 were assessed in 42 newly diagnosed CML-CP patients and 15 CML-BP patients. Quantitative real-time PCR, Western blots, and cell proliferation assay were used to characterize the changes induced by overexpression or inhibition of miR-181d, RBP2 or p65. Luciferase reporter assay and ChIP assay was conducted to establish functional association between miR-181d, RBP2 and p65. Inhibition of miR-181d expression and its consequences in tumor growth was demonstrated in vivo models.ResultsWe found that miR-181d was overexpressed in CML-BP, which promoted leukemia cell proliferation. Histone demethylase RBP2 was identified as a direct target of miR-181d which downregulated RBP2 expression. Moreover, RBP2 inhibited transcriptional expression of NF-κB subunit, p65 by binding to its promoter and demethylating the tri/dimethylated H3K4 region in the p65 promoter locus. In turn, p65 directly bound to miR-181d promoter and upregulated its expression. Therefore, RBP2 inhibition resulting from miR-181d overexpression led to p65 upregulation which further forwarded miR-181d expression. This miR-181d/RBP2/p65 feedback regulation caused sustained NF-κB activation, which contributed to the development of CML-BP.ConclusionsTaken together, the miR-181d/RBP2/p65 feedback regulation promoted CML-BP and miR-181d may serve as a potential therapeutic target of CML-BP.

Sirt-1 Regulates Physiological Process and Exerts Protective Effects against Oxidative Stress.

Background Recent studies suggest a correlation between the reduced Sirt-1 expression with Alzheimer's diseases (AD) and depression, respectively, suggesting a possible pathogenic role of the altered Sirt-1 expression in neuronal degenerative diseases, such as AD and depression. However, the molecular mechanisms underlying how Sirt-1 reduction impairs neuronal functions remain unknown. Methods We used the SK-N-SH neuroblastoma cells to study the role of Sirt-1 expression on physiological roles in neuronal cells. Gain of Sirt-1 was achieved by transiently transfecting Sirt-1 expression plasmid. Sirt-1-specific shRNA was used to elucidate the role of Sirt-1 loss of function. CCK-8 (Cell Counting Kit-8) assay and flow cytometry were used to evaluate cell proliferation. Semiquantitative western blotting was used to detect relative protein levels. A further luciferase reporter gene assay was employed to examine the effect of Sirt-1 expression on the transcriptional activity of p53. RT-qPCR was used to determine the mRNA levels of p21, Bax, and Bcl-2, which were the downstream target genes of p53. Results Sirt-1 suppressed the p53 downstream gene p21 transcription, while shRNA-mediated Sirt-1 knockdown resulted in a significant increase in p21 expression, implying a possibility that Sirt-1 promotes neuron proliferation through suppressing p53 transcriptional activity. The mRNA and protein levels of p53 were not affected by the altered Sirt-1 expression, suggesting that Sirt-1 regulates the transcriptional regulatory activity of p53 rather than p53 expression. Indeed, we further confirmed that Sirt-1 appeared to inhibit p53 transcriptional activity by attenuating its acetylation and resulted in a decrease of p53's binding to the p21 promoter. Overexpressed Sirt-1 scavenged reactive oxygen species (ROS) production in SK-N-SH with H2O2. Knockdown of Sirt-1 presented opposite effect; the addition of EX527 (Sirt-1 inhibitor) increased ROS accumulation. Conclusions Oxidative stress induces Sirt-1 in neuron cells, and Sirt-1 promotes proliferation in SK-N-SH cells, which protects them from oxidative stress-induced cell death, potentially via suppressing the transcriptional activity of p53. These results provide a molecular explanation underlying how the reduced Sirt-1 potentially causes the AD and depression-related diseases, supporting the idea that Sirt-1 can possibly be used as a diagnostic biomarker and/or therapeutic drug target for the AD and depression-related diseases.

Anaplastic Thyroid Carcinoma: Current Issues in Genomics and Therapeutics.

Anaplastic thyroid carcinoma is a type of thyroid carcinoma with the most aggressive biological behaviour amongst thyroid cancer. Here, we review the current genomic and the impacts of advances in therapies to improve the management of patients with the cancer. Common mutations being identified in anaplastic thyroid carcinoma are p53 and TERT promoter mutations. Other common mutated genes included BRAF, RAS, EIF1AX, PIK3CA, PTEN and AKT1, SWI/SNF, ALK and CDKN2A. Changes in expression of different microRNAs are also involved in the pathogenesis of anaplastic thyroid carcinoma. Curative resection combined with radiotherapy and combination chemotherapies (such as anthracyclines, platins and taxanes) has been shown to have effects in the treatment of some patients with anaplastic thyroid carcinoma. Newer molecular targeted therapies in clinical trials target mostly the cell membrane kinase and downstream proteins. These include targeting the EGFR, FGFR, VEGFR, c-kit, PDGFR and RET on the cell membrane as well as VEGF itself and the downstream targets such as BRAF, MEK and mTOR. Immunotherapy is also being tested in the cancer. Updated knowledge of genomic as well as clinical trials on novel therapies is needed to improve the management of the patients with this aggressive cancer.

Senescence associated long non-coding RNA 1 regulates cigarette smoke-induced senescence of type II alveolar epithelial cells through sirtuin-1 signaling

ObjectiveThe primary aim of our study was to explore the mechanisms through which long non-coding RNA (lncRNA)-mediated sirtuin-1 (SIRT1) signaling regulates type II alveolar epithelial cell (AECII) senescence induced by a cigarette smoke-media suspension (CSM).MethodsPharmacological SIRT1 activation was induced using SRT2104 and senescence-associated lncRNA 1 (SAL-RNA1) was overexpressed. The expression of SIRT1, FOXO3a, p53, p21, MMP-9, and TIMP-1 in different groups was detected by qRT-PCR and Western blotting; the activity of SA-β gal was detected by staining; the binding of SIRT1 to FOXO3a and p53 gene transcription promoters was detected by Chip.ResultsWe found that CSM increased AECII senescence, while SAL-RNA1 overexpression and SIRT1 activation significantly decreased levels of AECII senescence induced by CSM. Using chromatin immunoprecipitation, we found that SIRT1 bound differentially to transcriptional complexes on the FOXO3a and p53 promoters.ConclusionOur results suggested that lncRNA-SAL1-mediated SIRT1 signaling reduces senescence of AECIIs induced by CSM. These findings suggest a new therapeutic target to limit the irreversible apoptosis of lung epithelial cells in COPD patients.

The transcription factor PBX3 promotes tumor cell growth through transcriptional suppression of the tumor suppressor p53.

Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family and contains a highly conserved homologous domain. PBX3 is involved in the progression of gastric cancer, colorectal cancer, and prostate cancer; however, the detailed mechanism by which it promotes tumor growth remains to be elucidated. Here, we found that PBX3 silencing induces the expression of the cell cycle regulator p21, leading to an increase in colorectal cancer (CRC) cell apoptosis as well as suppression of proliferation and colony formation. Furthermore, we found that PBX3 is highly expressed in clinical CRC patients, in whom p21 expression is aberrantly low. We found that the regulation of p21 transcription by PBX3 occurs through the upstream regulator of p21, the tumor suppressor p53, as PBX3 binds to the p53 promoter and suppresses its transcriptional activity. Finally, we revealed that PBX3 regulates tumor growth through regulation of the p53/p21 axis. Taken together, our results not only describe a novel mechanism regarding PBX3-mediated regulation of tumor growth but also provide new insights into the regulatory mechanism of the tumor suppressor p53.

Curcumin ameliorates IL‐1β‐induced apoptosis by activating autophagy and inhibiting the NF‐κB signaling pathway in rat primary articular chondrocytes

Articular cartilage damage and chondrocyte apoptosis are common features of rheumatoid arthritis and osteoarthritis. Recently, curcumin has been reported to exhibit protective effects on degeneration in articular cartilage diseases. However, the effects and mechanisms of curcumin on articular chondrocyte injury remain to be elucidated. The aim of the present study is to investigate the chondroprotective mechanisms of curcumin on interleukin-1β (IL-1β)-induced chondrocyte apoptosis in vitro. The results revealed that IL-1β decreased cell viability and induced apoptosis in primary articular chondrocytes. Curcumin pretreatment reduced IL-1β-induced articular chondrocyte apoptosis. In addition, treatment with curcumin increased autophagy in articular chondrocytes and protected against IL-1β-induced apoptosis. The curcumin-mediated protection against IL-1β induced apoptosis was abolished when cells were treated with the autophagy inhibitor 3-methyladenine or transfected with Beclin-1 small interfering RNA. Furthermore, IL-1β stimulation significantly increased the phosphorylation levels of nuclear factor (NF)-κB p65 and glycogen synthase kinase-3β, and decreased the phosphorylation levels of β-catenin in articular chondrocytes, and these alterations to the phosphorylation levels were partly reversed by treatment with curcumin. Dual-luciferase and electrophoretic mobility shift assays demonstrated that IL-1β increased NF-κB p65 promoter activity in chondrocytes, and this was also reversed by curcumin. Pretreatment with the NF-κB inhibitor pyrrolidine dithiocarbamate enhanced the protective effects of curcumin on chondrocyte apoptosis, but Wnt/β-catenin inhibitor, XAV-939, did not exhibit this effect. Molecular docking and dynamic simulation studies results showed that curcumin could bound to RelA (p65) protein. These results indicate that curcumin may suppress IL-1β-induced chondrocyte apoptosis through activating autophagy and restraining NF-κB signaling pathway.

HOXA5 counteracts the function of pathological scar-derived fibroblasts by partially activating p53 signaling

The inactivation of p53 can lead to the formation of pathological scars, including hypertrophic scars and keloids. HOXA5 has been reported to be a critical transcription factor in the p53 pathway in cancers. However, whether HOXA5 also plays a role in pathological scar progression through activating p53 signaling remains unknown. In this study, we first demonstrated that HOXA5 overexpression in hypertrophic scar-or keloids-derived fibroblasts decreased cell proliferation, migration and collagen synthesis, whereas increased cell apoptosis. Furthermore, the results of luciferase activity assays and ChIP PCR assays indicated that HOXA5 transactivated p53 by binding to the ATTA-rich core motif in the p53 promoter. HOXA5 also increased the levels of p21 and Mdm2, which are downstream targets of p53. Interestingly, silencing p53 in these pathological scar-derived fibroblasts partially attenuated HOXA5-mediated growth inhibition effect and HOXA5-induced apoptosis. In addition, 9-cis-retinoic acid augmented the expression of HOXA5 and promoted the effects of HOXA5 on pathological scar-derived fibroblasts, and these effects could be suppressed by HOXA5 knockdown. Thus, our study reveals a role of HOXA5 in mediating the cellular processes of pathological scar-derived fibroblasts by transcriptionally activating the p53 signaling pathway, and 9-cis-retinoic acid may be a potential therapy for pathological scars.

HOTAIR promotes gefitinib resistance through modification of EZH2 and silencing p16 and p21 in non-small cell lung cancer

The long non-coding RNA Hox transcript antisense intergenic RNA (HOTAIR) plays a critical role in tumorigenesis as well as drug resistance in various cancers. However, the molecular mechanism by which HOTAIR induces gefitinib resistance in non-small cell lung cancer is to date unclear. In the present study, we revealed that HOTAIR is upregulated in gefitinib-resistant lung cancer cells and over-expression of HOTAIR enhances gefitinib resistance in lung cancer cells. In addition, the overexpression of HOTAIR promotes cell cycle progression through epigenetic regulation of EZH2/H3K27. Silencing of EZH2 by either siRNA or inhibitors sensitized the lung cancer cells to gefitinib. Inhibition of EZH2 induces expression of p16 and p21, whereas levels of CDK4, cyclinD1, E2F1, and LSD1 are significantly decreased in PC-9 cells overexpressing HOTAIR. ChIP-PCR experiments indicate that HOTAIR increases H3K27me3 recruitment to the promoter of p16 and p21 in PC-9 lung cancer cells overexpressing HOTAIR. In xenograft mouse models, overexpressing HOTAIR in lung cancer tissues decreased p16 and p21 proteins. Taken together, these data suggest that HOTAIR contributes to gefitinib resistance by regulating EZH2 and p16 and p21. Targeting HOTAIR may be a novel therapeutic strategy for treating gefitinib-resistance in non-small cell lung cancer.

Diagnostic roles of proliferative markers in pathological Grade of T1 Urothelial Bladder Cancer.

The stage T1 urothelial bladder cancer (T1 UBC) tumor grade classification is important for prognosis and clinical management. However, the reproducibility of this two-grade classification system is limited in regards to pathological diagnosis, and there is lack of ideal, objective and easily detected markers for pathological diagnosis. In our study, bladder urothelial lesions from a total of 124 patients diagnosed pathologically after transurethral resection of the bladder tumor (TURBT) were collected, including non-cancerous lesions from 33 patients and lesions from 91 T1 UBC patients. A series of previous studies have suggested some common and valuable factors in the diagnosis and prognosis of UBC, but there are still some controversial factors, such as the mitotic figure (MF) of tumor cell, cell proliferation index Ki-67, graded differentiation marker CK20, P53, P504S and carcinogenesis associated telomerase reverse transcriptase (TERT) promoter mutations. The purpose of this study was to evaluate the value of these factors in the pathological grading diagnosis of T1 UBC. The results showed that gender, lesion size, mitotic index (MI), CK20, P53, Ki-67, P504S and TERT promoter hot spot mutations (C228T and C250T) were correlated with T1 UBC diagnosis (P<0.05). The MI, Ki-67 and P504S were correlated with the pathological grade of T1 UBC (P<0.05). Logistic regression analysis showed that the MI and Ki-67 were independent risk factors for high-grade (HG) of T1 UBC (P<0.05). The combined detection of the MI, Ki-67 and P504S in a multivariate diagnostic model improved the diagnostic accuracy of assigning the T1 UBC pathological grade (AUC=0.904, 95%CI: 0.824~0.956, P<0.05). In conclusion, MI and Ki-67, as important markers of histopathology and cell proliferation, can be easily measured and have good reproducibility. These markers may be meaningful parameters for assigning the pathological grade of UBC.

The Differential Antitumor Activity of 5-Aza-2'-deoxycytidine in Prostate Cancer DU145, 22RV1, and LNCaP Cells.

DNA methylation is a DNA methyltransferase-mediated epigenetic modification affecting gene expression. This process is involved in the initiation and development of malignant disease. 5‐Aza‐2′‐deoxycytidine (5‐Aza), a classic DNA methyltransferase inhibitor, possesses antitumor proliferation activity. However, whether 5-Aza induces cytotoxicity in solid tumors warrants further investigated. In this study, human prostate cancer (CaP) cells were treated with 5-Aza and subjected to cell viability and cytotoxicity analysis. Reverse transcription-polymerase chain reaction and methylation-specific polymerase chain reaction assay were utilized to test the gene expression and methylation status of the p53 and p21 gene promoters. The results showed that 5-Aza differentially inhibited spontaneous proliferation, arrested the cell cycle at S phase in DU145, at G1 phase in 22RV1 and LNCaP cells, and G2 phase in normal RWPE-1 cells, as well as induced the expression of phospho-H2A.X and tumor suppressive mammary serine protease inhibitor (maspin) in all three types of CaP cells. 5-Aza also increased p53 and p21 transcription through promoter demethylation, and decreased the expression of oncogene c-Myc in 22RV1 and LNCaP cells. Western blotting analysis showed that the poly (ADP-ribose) polymerase cleavage was detected in DU145 and 22RV1 cells. Moreover, there were no significant changes in p53, p21 and c-Myc expression in DU145 cells following treatment with 5-Aza. Thus, in responsible for its apoptotic induction and DNA damage, the mechanism of the antitumor activities of 5-Aza may involve in an increase of tumor suppressive maspin, upregulation of wild type p53-mediated p21 expression and a decrease of oncogene c-Myc level in 22RV1 and LNCaP cells, and enhancing the tumor suppressive maspin expression in DU145 cells. These results enriched our understanding of the multifaceted antitumor activity of 5-Aza, and provided the expression basis of biomarkers for its possible clinical application in prostate cancer.