Negative Regulator Of Cell Proliferation Research Articles

Circular RNA expression profile and potential function of hsa_circRNA_101238 in human thoracic aortic dissection.

ObjectiveTo assess the circular RNAs (circRNAs) expression profile and explore the potential functions in human thoracic aortic dissection (TAD).MethodsThe differentially expressed circRNAs profiles of the aortic segments between human type A TAD patients (n=3) and age-matched normal donors (NA; n=3) were analyzed using the Arraystar human circRNAs microarray. Quantitative real-time PCR was used to validate the expression pattern of circRNAs, parental genes, and hsa-miR-320a; Western blotting confirmed MMP9 expression with additional samples. Bioinformatic tools including network analysis, Gene ontology, and KEGG pathway analysis were utilized.ResultsAmong 8,173 detected circRNA genes, 156 upregulated and 106 downregulated significantly in human TAD as compared to NA (P£0.05). Quantitative real-time PCR showed an elevated expression of the upregulated hsa_circRNA_101238, hsa_circRNA_104634, hsa_circRNA_002271, hsa_circRNA_102771, hsa_circRNA_104349, COL1A1, and COL6A3 and reduced of the downregulated hsa_circRNA_102683, hsa_circRNA_005525, hsa_circRNA_103458, and FLNA. Gene ontology analysis revealed that the parental genes favored several pathological processes, such as negative regulation of cell proliferation and extracellular matrix organization. The circRNA-miRNA co-expression network predicted that 33 circRNAs might interact with at least one target miRNAs altered in TAD. KEGG pathway analysis revealed that 28 altered miRNAs were enriched on focal adhesion and vascular smooth muscle contraction. The hsa_circRNA_101238-miRNA-mRNA network indicated the highest degree of hsa-miR-320a. Quantitative real-time PCR and Western blot manifested the low expression of hsa-miR-320a and high of MMP9 in human TAD tissues, respectively.ConclusionsThis study revealed hundreds of differentially expressed circular RNAs in human TAD, suggesting that hsa_circRNA_101238 might inhibit the expression of hsa-miR-320a and increase that of MMP9 in TAD.

Abstract 5756: Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro

Abstract Infection by the Epstein-Barr virus (EBV) is associated with a variety of human cancers, notably Burkitt lymphoma and nasopharyngeal carcinoma (NPC). Most malignant cells within EBV-associated cancers are latently infected, and some viral products expressed during the latent phase of the viral cycle have well-described carcinogenicity, including the EBV latent membrane protein 1 (LMP1). Distinct viral genotypes described so far include the B95.8, the most studied viral strain originally found in an elderly patient with transfusion-induced infectious mononucleosis, and the M81 strain, isolated from a NPC case from a Chinese patient. Compared to B95.8, the EBV M81 strain infects epithelial cells more readily, and it is more likely to induce spontaneous viral lytic reactivation. However, whether these EBV strains have distinct biological features relevant for the progression of EBV-associated cancers remains to be elucidated. Thus, this study aimed to evaluate whether the expression of B95.1 and M81 variants of EBV LMP1 have different impact on cell migration and invasiveness of human cells lines in vitro, as well as on the expression of selected endogenous microRNAs (miRs) that were previously reported to have a role on cancer progression. Expression of EBV LMP1 was achieved in 293 and NP69 cells transfected with vectors encoding its B95.8 or M81 variants. In vitro migration and invasion rates were assessed by the scratch wound healing assay and the Boyden chamber assay, respectively. The expression of 91 selected miRs were evaluated by a customized qPCR-array approach. Cells expressing EBV LMP1 showed increased migration and invasion rates compared with mock-transfected cells. However, the differences observed between B95.8 and M81 variants did not achieve statistical significance. Conversely, 293 cells expressing the M81 variant of LMP1 showed hyperexpression of human miRs 497-5p, 17-3p, and 34a-5p, compared to B95.8. The in silico target prediction for these miRs showed enrichment of gene targets involved in the MAPK, NF-κB, and PI3K/AKT intracellular signaling pathways. This would reflect in negative regulation of cell proliferation and cell motility/adhesion, as well as higher levels of apoptosis. Based on these data, it is plausible to suggest that EBV LMP1 from M81 viral strain is somewhat less effective in terms of carcinogenicity, and this might be a reflect of the improved ability of EBV M81 to induce lytic viral infection compared to the B95.8 strain, as previously reported. In conclusion, the results indicate that cells expressing LMP1 from EBV isolates B95.8 and M81 showed differences in terms of miR expression signatures, which ultimately might impact the pathogenesis and progression of EBV-associated cancers. Citation Format: Bárbara Grasiele Müller-Coan, Ethel Cesarman, Deilson Elgui De Oliveira. Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5756. doi:10.1158/1538-7445.AM2017-5756

Abstract 2112: FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells

Abstract Pancreatic cancer is highly chemo-resistant associated with oncogenic mutations such as KRAS and/or p53. The effect of conventional chemotherapy is not sufficient and new target and strategy is urgently needed. The forkhead box (Fox) proteins are multidirectional transcriptional factors strongly implicated in malignancies. Although Fox O (FoxO) protein, and particularly FoxO3a, works as negative regulator of cell proliferation by repressing cyclin proteins and inducing cell cycle inhibitors such as p21Waf1/Cip1, its expression is consistently suppressed by several oncogenic pathways including phosphatidylinositol-3 kinase (PI3K) / AKT pathway, constitutively activated in pancreatic cancer. Thus, upregulating FoxO3a activity could be a promising target of pancreatic cancer treatment without impact of underlying oncogenic mutations. Class IIa Histone deacetylase (HDAC) is a subgroup of HDAC. Though HDAC inhibitors (HDACi) have been extensively investigated as a cancer target, its action mechanism is considered due to histone modification by class I. Biological significance of class IIa HDACs which have minimal histone deacetylation activity have not been elucidated yet. Recent studies show class IIa HDACs act as a transcriptional regulator including FoxO3a. In this study, we investigate the biologic impact of HDAC class IIa inhibition on FoxO3a and anti-tumor effect against pancreatic cancer cell line using selective class IIa HDACi TMP269. TMP269 treatment showed increased FoxO3a expression in a dose dependent manner with immunoblotting and modest cell growth inhibition effect at 57.5 μM of IC50 dose for 48-hour treatment against AsPC-1 in MTT. G1/S arrest was observed with cell cycle assay. Upregulated p21Waf1/Cip1 and downregulated CDK2 and 4/6 and cyclin D1 and D2 expressions were further observed, consistent with inducing G1/S arrest and transcriptionally activated FoxO3a. Importantly, upregulated p21Waf1/Cip1 was observed in AsPC-1 p53 null cell line, suggesting independent with p53 pathway. These findings suggest upregulated FoxO3a induced by HDAC class IIa inhibition activated its transcription and resulted in cell growth inhibition. Because PI3K/AKT leads FoxO3a to the ubiquitylation-mediated proteasome degradation, we examined irreversible proteasome inhibitor carfilzomib (CFZ) combined with TMP269, aiming synergistic FoxO3a upregulating. As expected, FoxO3a expression was further increased in TMP269 combined with CFZ compared with TMP269 or CFZ alone. Following the activated FoxO3a, p21Waf1/Cip1 expression was upregulated and cell growth inhibition was dramatically enhanced. In conclusion, HDAC class IIa inhibition modified FoxO3a transcriptional activation and upregulating FoxO3 by dual inhibition of HDAC class IIa and proteasome is promising target against pancreas cancer. Citation Format: Makoto Usami, Shohei Kikuchi, Kohichi Takada, Yusuke Sugama, Yohei Arihara, Naotaka Hayasaka, Hajime Nakamura, Yuki Ikeda, Yusuke Kamihara, Masahiro Hirakawa, Makoto Yoshida, Masayoshi Kobune, Koji Miyanishi, Junji Kato. FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2017-2112

Review: Biological and Molecular Differences between Tail Regeneration and Limb Scarring in Lizard: An Inspiring Model Addressing Limb Regeneration in Amniotes.

Tissue regeneration in lizards represents a unique model of regeneration and scarring in amniotes. The tail and limb contain putative stem cells but also dedifferentiating cells contribute to regeneration. Following tail amputation, inflammation is low and cell proliferation high, leading to regeneration while the intense inflammation in the limb leads to low proliferation and scarring. FGFs stimulate tail and limb regeneration and are present in the wound epidermis and blastema while they disappear in the limb wound epidermis 2-3 weeks postamputation in the scarring outgrowth. FGFs localize in the tail blastema and the apical epidermal peg (AEP), an epidermal microregion that allows tail growth but is absent in the limb. Inflammatory cells invade the limb blastema and wound epidermis, impeding the formation of an AEP. An embryonic program of growth is activated in the tail, dominated by Wnt-positive and -negative regulators of cell proliferation and noncoding RNAs, that represent the key regenerative genes. The balanced actions of these regulators likely impede the formation of a tumor in the tail tip. Genes for FACIT and fibrillar collagens, protease inhibitors, and embryonic keratins are upregulated in the regenerating tail blastema. A strong downregulation of genes for both B and T-lymphocyte activation suggests the regenerating tail blastema is a temporal immune-tolerated organ, whereas a scarring program is activated in the limb. Wnt inhibitors, pro-inflammatory genes, negative regulators of cell proliferation, downregulation of myogenic genes, proteases, and oxidases favoring scarring are upregulated. The evolution of an efficient immune system may be the main limiting barrier for organ regeneration in amniotes, and the poor regeneration of mammals and birds is associated with the efficiency of their mature immune system. This does not tolerate embryonic antigens formed in reprogrammed embryonic cells (as for neoplastic cells) that are consequently eliminated impeding the regeneration of lost organs.

Identification of spinach SIAMESE and analysis of its function in plant immunity

Diseases cause a significant loss in both yield and quality of spinach. The cell cycle signalling pathway plays a central role in balancing development and immunity of plants. Cyclin-Dependent Kinase Inhibitor (CKI) is a core cell cycle regulator. It has been found that two Arabidopsis CKIs, SIAMESE (SIM) and SMR1 (SIAMESE-Related 1), function redundantly not only as negative regulators of cell proliferation but also as positive regulators of plant immunity. In the spinach genome, we identified a homologue of Arabidopsis SIM, referred to as Spinacia oleracea SIAMESE (SoSIM). To investigate the function of SoSIM, we introduced the 35S promoter-driven SoSIM (35S:SoSIM) into Arabidopsis sim smr1 double mutants. Over-expression of SoSIM phenocopied RNA interference of Cyclin-Dependent Kinase A1 (CDKA1) which exhibited dwarf and serrated leaves, confirming that SIM is a CDK inhibitor. Arabidopsis wild-type trichomes are single and unicellular, whereas sim smr1 mutant trichomes are clustered and multicellular. SoSIM restored wild-type trichome phenotype of sim smr1 mutant. The sim smr1 mutants were susceptible to the avirulent pathogen Pseudomonas syringae pv. maculicola as compared with wild-type plants. Our data showed that the resistance of 35S:SoSIM-transgenic sim smr1 lines to the pathogen was fully restored, indicating that SoSIM activates plant immunity. These data verify that SoSIM functions as its Arabidopsis counterpart in inhibition of plant development and activation of plant immunity. Therefore, SoSIM can be explored to control the balance between development and immunity in spinach.

Cholesterol loading rescues OxLDL‐induced activation of endothelial Rho/ROCK/Akt/p27kip1 cascade

Oxidized modifications of LDL (oxLDL) are known to activate multiple signaling pathways that lead to impairment and disbalance of endothelial function impairment of the barrier function and including abnormal proliferation. It is generally believed that oxLDL exerts its effects on endothelial function via enhanced loading of cholesterol. Our studies, however, show that this is not the case. In contrast, we show that oxLDL and cholesterol loading have opposite effects on several key signaling events in human aortic endothelial cells (HAECs). Moreover, we show that cholesterol loading may rescue oxLDL‐induced effects on EC signaling. We also we provide comparative analysis of the effects of two major types of oxLDL, copper oxidized LDL (Cu‐oxLDL) and Lipoxygenase oxidized LDL (LPO‐oxLDL). Specifically, we show that both, Cu‐oxLDL or LPO‐oxLDL significantly increase Akt phosphorylation whereas inhibiting Akt with a specific inhibitor MK2206 blocks oxLDL‐induced increase in the proliferation of HAECs. Furthermore, blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibits oxLDL‐induced Akt phosphorylation and cell proliferation induced by both Cu‐ and LPO‐oxLDL. In contrast, blocking Rac1 with MSC inhibitor has no effect neither on oxLDL‐induced Akt phosphorylation or cell proliferation of HAECs. Further evidence shows that oxLDL‐induced Rho/Akt pathway leads to downregulation of p27kip1, a well‐known negative regulator of cell proliferation. Most importantly, we also show that preloading cells with cholesterol prevents oxLDL‐induced activation of RhoA and oxLDL‐induced Akt phosphorylation. Furthermore, cholesterol pre‐loading also rescues oxLDL‐induced loss of p27kip1 expression. We propose that oxLDL impairs endothelial function by increasing the cellular content of oxysterols which disrupt the structure of endothelial membrane, an effect that can be repaired by an increase in cellular cholesterol.Support or Funding InformationNIH grants HL‐073965 and HL‐083298 to I.L.

A histone deacetylase inhibitor enhances expression of genes inhibiting Wnt pathway and augments activity of DNA demethylation reagent against nonsmall-cell lung cancer

Development of new inhibitors targeting histone deacetylases (HDACs) with improved efficacy for solid tumor therapy is urgently needed. Here, we report the development of a novel HDAC inhibitor TMU-35435 and verify it as a single agent and in combination treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) in lung cancer preclinical models. TMU-35435 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. Expression microarrays revealed a unique TMU-35435-induced gene networks enriched in biological processes, including "negative regulation of cell proliferation" and "Wnt receptor signaling pathway" compared to FDA-approved HDAC inhibitor SAHA. TMU-35435 inhibited tumor growth with good pharmacokinetic properties and safety features in lung orthotopic and subcutaneously implanted xenograft models. TMU-35435 and 5-aza-dC showed synergistic antitumor effects through reactivation of tumor suppressor genes and those genes encoding negative regulators of Wnt signaling pathway in vitro and in vivo. Some genes showed additive inhibition of DNA methylation upon TMU-35435 and 5-aza-dC combined treatment. Our findings suggested that TMU-35435 is a potential HDAC inhibitor for lung cancer treatment as a single agent and in combination with 5-aza-dC.

P27Kip1 promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway

p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Raslox (H-Ras -/-, N-Ras -/-, K-Ras lox/lox, RERTert/ert) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Morphine Suppresses Lung Cancer Cell Proliferation Through the Interaction with Opioid Growth Factor Receptor: An In Vitro and Human Lung Tissue Study.

There have been inconsistent reports on whether opioids promote or inhibit lung cancer growth. In this study, we suggest that opioid growth factor receptor (OGFR), a negative regulator of cell proliferation, is a binding site of morphine and is involved in subsequent morphine-induced lung cancer growth suppression. The expression and distribution of OGFR in human lung cancer tissues and cell lines were assessed with immunohistochemistry and real-time reverse transcription polymerase chain reaction. The human lung cancer cell line, H1975 (adenocarcinoma), which overexpressed OGFR but not μ-opioid receptors, was selected for further analysis to verify the interaction between morphine and OGFR and the impact of morphine on cancer cell growth. OGFR was expressed in lung cancer tissues and all cancer cell lines tested. Adenocarcinoma showed a higher OGFR expression than squamous cell carcinoma (reverse transcription polymerase chain reaction relative quantitation value: median [interquartile range], 13.1 [9.3-20.0] vs 4.3 [2.2-6.6]; P = 0.003). OGFR expression showed an inverse correlation with cell proliferation (r = -0.92, P = 0.0001). Morphine treatment reduced the median H1975 cell number by approximately 23% (P = 0.03). Growth suppression by morphine was attenuated when OGFR was knocked down. A confocal experiment demonstrated binding of morphine to OGFR. Growth suppression by morphine occurred in the S phase of the cell cycle. Lung cancer tissues and cell lines express OGFR. Morphine interacts with OGFR and may suppress lung cancer progression.

5α-Dihydrotestosterone negatively regulates cell proliferation of the periurethral ventral mesenchyme during urethral tube formation in the murine male genital tubercle.

Androgen is an essential factor involved in masculinization of external genitalia. Failure of the exposure to 5α-dihydrotestosterone (DHT) causes a hypoplastic penile size and urethral abnormality. The main pathology of hypospadias is defective urethral closure on the ventral side of the penis. Hormone-dependent genes are suggested as the causative factors. However, the detailed mechanisms of DHT functions on urethral tube formation remain unknown. Androgen is both a positive and negative regulator of cell proliferation. The roles of locally converted DHT in cell proliferation at the periurethral mesenchyme have not been elucidated. We revealed the expression pattern of 5α-reductase type 2 mRNA (Srd5a2) and local DHT distribution by direct measurement in this study. We also analyzed periurethral mesenchymal cell proliferation status using systematic three-dimensional (3D) reconstruction analyses. A prominent Srd5a2 expression and localized DHT distribution on the ventral side of the genital tubercle were detected. Cell proliferation was reduced in this mesenchymal region during urethral formation. The current results suggest the presence of the possible negative regulation of cell proliferation by DHT. Moreover, cell proliferation related to urethral tube formation was revealed to be DHT dose dependent. These data are expected to contribute to the understanding of the mode of regulation of cell proliferation related to urethral tube formation by DHT. These findings may also offer insight into the understanding of human hypospadias and related hormone-dependent factors.

Abstract 2778: Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma

Abstract Introduction: Colorectal Cancer (CRC) is one of the most common malignancies worldwide. The role of MSI and KRAS somatic mutation in tumor tissue is well known in CRC. In genome-wide scale, we explored whether differential methylation is associated with MSI status. Methods: We carried out a genome-wide methylation assay (Illumina 450K) for a total of 250 paired samples from 125 CRC patients (m = 72, f = 53) at different stages (stage1:25, stage II: 33 and stageIII: 67). Of them 101 had left-sided (descending colon to rectum) CRC and 30 had MSI, and 34 had somatic mutation in KRAS (rs112445441). Results: MSI was more frequent in the right-sided tumor (54% vs. 17%, p = 0.003). Frequency of KRAS mutation was not different between right and left-sided CRC (29% vs. 27%). Among the patients with microsatellite stable (MSS) CRC (n = 95), paired comparison of methylation data between tumor and corresponding normal tissue revealed a total of 1641 tumor-specific differentially methylated loci (DML) covering 686 genes that were significant at FDR 0.001 and the magnitude of difference (delta beta) was at least 20%; Similar analysis in patients with MSI (n = 30) revealed 6209 tumor-specific DML covering 2316 genes. This suggested that MSI is associated with methylation change in much larger number of genes. We could not find any methylation signature from normal colon tissue that could predict MSI or KRAS mutation in the corresponding tumor tissue. However, we identified 413 genes to be differentially methylated in tumor tissue compared to corresponding normal tissue only in presence of MSI, irrespective of KRAS mutation status, tumor staging, and location of tumor. These are “MSI-associated tumor-specific genes”. Among these, 19 DML covering17 genes showed delta-beta >30%. The list was enriched in genes associated with biologically relevant GO-terms like, “negative regulation of cell proliferation”, “regulation of MAP kinase activity”, “regulation of biological process” etc. We also identified 240 genes differentially methylated in tumor tissue compared to corresponding normal tissue irrespective of MSI status, KRAS mutation, tumor staging, and location of tumor. These are “general tumor-specific genes”. Among these loci, 87 DML (49 genes) showed delta-beta >30%. Conclusions: Our study shows evidence of association between MSI and DNA methylation in the pathogenesis of CRC. Citation Format: Muhammad G. Kibriya, Mohammed Kamal, Mustafizur Rahman, Shantanu Roy, Maruf Raza, Rupash Paul, Habibul Ahsan, Zahidul Haq, Farzana Jasmine. Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2778.

Abstract 2924: Metastasis-associated protein 1 induces tamoxifen resistance in MCF7 breast cancer cells

Abstract Tamoxifen is most commonly used to treat estrogen receptor-alpha positive breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Metastasis associated protein 1 (MTA1) is a cancer progression-related gene and has been reported to be overexpressed in a variety of human cancers including breast cancer. Here, we showed that transient overexpression of MTA1 in MCF7 breast cancer cells results in tamoxifen resistance, whereas si-RNA mediated knockdown of MTA1 was more susceptible to tamoxifen-induced growth inhibition. Moreover, stable overexpression of MTA1 in MCF7 cells promoted cell proliferation and reduced tamoxifen sensitivity. To explore the mechanisms of MTA1-mediate tamoxifen resistance, we used microarray analysis to profile the gene expression patterns of MTA1 overexpress MCF7 stable cells after tamoxifen treatment. From microarray data analysis, 538 genes were selected as associated with MTA1-mediated tamoxifen resistance (P < 0.05, LPE test; cutoff ≥ 1.5-fold). The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis revealed that the predominant biological processes associated with these genes are M phase, negative regulation of cell proliferation, mitosis and M phase of mitotic cell cycle. Further, we found that cell-cycle regulator Cyclin D1 and cyclin B1 protein levels were increased in MTA1 overexpression MCF7 stable cells. Analyses of publicly available patient data sets indicated that MTA1 levels are higher in patients after endocrine therapy failure (recurrence), compared to those of responders (non-recurrence). These results suggest that MTA1 may contributed tamoxifen resistance through the regulation of cell cycle components. Citation Format: Min-Ho Lee, Dahae Koh, Mi-Ock Lee. Metastasis-associated protein 1 induces tamoxifen resistance in MCF7 breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2924.

RB1: a prototype tumor suppressor and an enigma.

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene to be molecularly defined. RB1 mutations occur in almost all familial and sporadic forms of retinoblastoma, and this gene is mutated at variable frequencies in a variety of other human cancers. Because of its early discovery, the recessive nature of RB1 mutations, and its frequency of inactivation, RB1 is often described as a prototype for the class of tumor suppressor genes. Its gene product (pRB) regulates transcription and is a negative regulator of cell proliferation. Although these general features are well established, a precise description of pRB's mechanism of action has remained elusive. Indeed, in many regards, pRB remains an enigma. This review summarizes some recent developments in pRB research and focuses on progress toward answers for the three fundamental questions that sit at the heart of the pRB literature: What does pRB do? How does the inactivation of RB change the cell? How can our knowledge of RB function be exploited to provide better treatment for cancer patients?

Cohesin subunits, STAG1 and STAG2, and cohesin regulatory factor, PDS5b, in oral squamous cells carcinomas

Cohesin complex is responsible for sister chromatid cohesion. STAG1/STAG2 is part of the complex, which is regulated by PDS5B. Alterations in these genes were described in tumors. PDS5B is a negative regulator of cell proliferation. We aimed to assess molecular alterations in these genes in oral squamous cell carcinoma (OSCC) and predict their expression by the expression of 84 cell cycle genes. In addition, we investigated whether pds5b protein expression impacted ki-67 and p53 immunopositivity. We assessed loss of heterozygosity (LOH) at STAG1 and STAG2 loci in 15 OSCC using three polymorphic markers. Associations between the immunoexpression of pds5b and ki-67 and p53 were tested in 62 samples. Differences between transcriptional levels of STAG1, STAG2, and PDS5B between OSCC and normal oral mucosa (NM) were evaluated by qPCR. An 84 cell cycle genes qPCR array was carried with OSCC samples, and STAG1, STAG2, and PDS5B were independently used as response variables in multiple linear regression models. Loss of heterozygosity in at least one marker was observed in three samples. pds5b, p53, and ki-67 were highly expressed, and no association was found between pds5b immunoexpression and ki-67 or p53 (P > 0.05). OSCC and NM showed similar transcriptional levels of STAG1, STAG2, and PDS5B. STAG1 and CUL3 expression seem to be related (P = 0.004). There is LOH at STAG1 and STAG2 loci in OSCC, but OSCC and NM showed similar transcriptional levels of STAG1, STAG2, and PDS5B. pds5b immunoexpression in OSCC was high, but it was not associated with proliferation cell index.

Deletion and low expression of NFKBIA are associated with poor prognosis in lower-grade glioma patients.

Lower-grade gliomas (LGGs), which are uniformly fatal in young adults, are classified as grades II-III tumors according to their histological features. The NFκB transcription factor, a crucial player in cancer initiation and progression, is inactivated in the cytoplasm by inhibitory proteins (IκBs) that have been shown to exert tumor-suppressor activity. Therefore, using The Cancer Genome Atlas copy number alteration and RNA-Seq data from 398 patients, we evaluated the association between the expression and dosage of NFKBIA, which encodes IκBα, and the overall malignancy of LGGs. Deletion and low expression of NFKBIA were associated with enhanced tumor aggressiveness and poor prognosis in LGGs. Accordingly, the dosage and expression of NFKBIA were independent prognostic factors for 5-year survival (dosage: P = 0.016; expression: P = 0.002) and 5-year recurrence-free survival (dosage: P = 0.009; expression: P = 0.005). Moreover, gene set enrichment analyses and co-expression network analyses indicated a role for NFKBIA in the negative regulation of cell proliferation, possibly through the modulation of downstream NFκB activation. Overall, the present findings reveal the prognostic value of NFKBIA in LGGs, reinforcing the relevance of NFκB signaling in the development and progression of gliomas.

Transcriptome wide analyses reveal a sustained cellular stress response in the gill tissue of Trematomus bernacchii after acclimation to multiple stressors.

BackgroundAs global climate change progresses, the Southern Ocean surrounding Antarctica is poised to undergo potentially rapid and substantial changes in temperature and pCO2. To survive in this challenging environment, the highly cold adapted endemic fauna of these waters must demonstrate sufficient plasticity to accommodate these changing conditions or face inexorable decline. Previous studies of notothenioids have focused upon the short-term response to heat stress; and more recently the longer-term physiological response to the combined stress of increasing temperatures and pCO2. This inquiry explores the transcriptomic response of Trematomus bernacchii to increased temperatures and pCO2 at 7, 28 and 56 days, in an attempt to discern the innate plasticity of T. bernacchii available to cope with a changing Southern Ocean.ResultsDifferential gene expression analysis supported previous research in that T. bernacchii exhibits no inducible heat shock response to stress conditions. However, T. bernacchii did demonstrate a strong stress response to the multi-stressor condition in the form of metabolic shifts, DNA damage repair, immune system processes, and activation of apoptotic pathways combined with negative regulation of cell proliferation. This response declined in magnitude over time, but aspects of this response remained detectable throughout the acclimation period.ConclusionsWhen exposed to the multi-stressor condition, T. bernacchii demonstrates a cellular stress response that persists for a minimum of 7 days before returning to near basal levels of expression at longer acclimation times. However, subtle changes in expression persist in fish acclimated for 56 days that may significantly affect the fitness T. bernacchii over time.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2454-3) contains supplementary material, which is available to authorized users.

Behavioral abnormalities in mice lacking mesenchyme-specific Pten

Phosphatase and tensin homolog (Pten) is a negative regulator of cell proliferation and growth. Using a Cre-recombinase approach with Lox sequences flanking the fibroblast-specific protein 1 (Fsp1 aka S100A4; a mesenchymal marker), we probed sites of expression using a β-galactosidase Rosa26LoxP reporter allele; the transgene driving deletion of Pten (exons 4–5) was found throughout the brain parenchyma and pituitary, suggesting that deletion of Pten in Fsp1-positive cells may influence behavior. Because CNS-specific deletion of Pten influences social and anxiety-like behaviors and S100A4 is expressed in astrocytes, we predicted that loss of Pten in Fsp1-expressing cells would result in deficits in social interaction and increased anxiety. We further predicted that environmental enrichment would compensate for genetic deficits in these behaviors. We conducted a battery of behavioral assays on Fsp1-Cre;PtenLoxP/LoxP male and female homozygous knockouts (Pten−/−) and compared their behavior to PtenLoxP/LoxP (Pten+/+) conspecifics. Despite extensive physical differences (including reduced hippocampal size) and deficits in sensorimotor function, Pten−/− mice behaved remarkably similar to control mice on nearly all behavioral tasks. These results suggest that the social and anxiety-like phenotypes observed in CNS-specific Pten−/− mice may depend on neuronal Pten, as lack of Pten in Fsp1-expressing cells of the CNS had little effect on these behaviors.

KDM2B/FBXL10 targets c-Fos for ubiquitylation and degradation in response to mitogenic stimulation

SUMMARYKDM2B (also known as FBXL10) controls stem cell self-renewal, somatic cell reprogramming and senescence, and tumorigenesis. KDM2B contains multiple functional domains, including a JmjC domain that catalyzes H3K36 demethylation and a CxxC zing finger that recognizes CpG islands and recruits the polycomb repressive complex 1 (PRC1). Here, we report that KDM2B, via its F-box domain, functions as a subunit of the CUL1-RING ubiquitin ligase (CRL1/SCFKDM2B) complex. KDM2B targets c-Fos for polyubiquitylation and regulates c-Fos protein levels. Unlike the phosphorylation of other SCF/CRL1 substrates that promotes substrates binding to F-box, EGF-induced c-Fos S374 phosphorylation dissociates c-Fos from KDM2B and stabilizes c-Fos protein. Non-phosphorylatable and phosphomimetic mutations at S374 result in c-Fos protein which cannot be induced by EGF and accumulates constitutively and lead to decreased or increased cell proliferation, respectively. Multiple tumor-derived KDM2B mutations impaired the function of KDM2B to target c-Fos degradation and to suppress cell proliferation. These results reveal a novel function of KDM2B in the negative regulation of cell proliferation by assembling an E3 ligase to targeting c-Fos protein degradation that is antagonized by mitogenic stimulations.

Suppression of trophoblast cell surface antigen 2 enhances proliferation and migration in liver fluke-associated cholangiocarcinoma

Background and aim. Trophoblast cell surface antigen 2 (TROP2) or tumor-associated calcium signal transducer 2 (TACSTD2) is a 36-kDa type I transmembrane glycoprotein and exerts dual functions as an oncogene and tumor suppressor in cancer cells. In this study, we investigated the expression and functions of TROP2 in liver fluke-associated cholangiocarcinoma (CCA).Material and methods. TROP2 expression in 85 CCA tissues was detected by using immunohistochemistry. The methylation status of TROP2 promoter was studied in 15 matched pairs of normal and CCA formalin fixed paraffin embedded (FFPE) tissues using the bisulfite genomic sequencing (BGS) method. The functions of TROP2 on cancer cell behavior were investigated using siRNA in CCA cell lines. Proliferation, migration and invasion assays were performed. A PCR array was used to evaluate the impact of TROP2 knockdown on the gene expression profiles.Results. TROP2 was highly expressed in all normal bile duct epithelia, but significantly down-regulated in CCA cells. Sixty percent of CCA revealed promoter hypermethylation compared to the corresponding adjacent normal tissues. TROP2 knockdown significantly enhanced the proliferation and migration in CCA cell lines, and altered the expressions of MARCK, EMP1and FILIP1L.Conclusion. We provide new evidence that TROP2 is epigenetically down-regulated and operates as a negative regulator of cell proliferation and migration in liver fluke-associated CCA.