Regulation Of Cyclin D1 Expression Research Articles

ERK1/2-dependent activation of mTOR/mTORC1/p70S6K regulates thrombin-induced RPE cell proliferation

Epithelial–mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-ζ/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1.

H2A.Z-dependent crosstalk between enhancer and promoter regulates Cyclin D1 expression

H2A.Z association with specific genomic loci is thought to contribute to a chromatin structure that promotes transcription activation. Acetylation of H2A.Z at promoters of oncogenes has been linked to tumorigenesis. The mechanism is unknown. Here, we show that in triple negative breast cancer cells, H2A.Z bound to the promoter of the constitutively, weakly expressed cyclin D1 oncogene (CCND1), a key regulator of cellular proliferation. Depleting the pool of H2A.Z stimulated transcription of CCND1 in the absence of its cognate transcription factor, the estrogen receptor (ER). During activation of CCND1, H2A.Z was released from the transcription start site (TSS) and downstream enhancer (enh2) sequences. Concurrently, acetylation of H2A.Z, H3 and H4 at the TSS was increased but only H2A.Z was acetylated at enh2. Acetylation of H2A.Z required the Tip60 acetyltransferase to be associated with the activated CCND1 on both TSS and enh2 sites. Depletion of Tip60 prevented CCND1 activation. Chromosome conformation capture experiments (3C) revealed specific contacts between the TSS and enh2 chromatin regions. These results suggest that release of a histone H2A.Z-mediated repression loop activates CCND1 for transcription. Our findings open new avenues for controlling and understanding aberrant gene expression associated with tumorigenesis.

Targeting Estrogen Receptor-α Reduces Adrenocortical Cancer (ACC) Cell Growthin Vitroandin Vivo: Potential Therapeutic Role of Selective Estrogen Receptor Modulators (SERMs) for ACC Treatment

Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. The objective of this study was to define the role played by ERα in 17β-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.

The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation

ObjectiveHepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.MethodsWe established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.ResultsHBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.ConclusionmiR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.

Cyclin D1 expression in podocytes: regulated by mitogens in collaboration with integrin–extracellular matrix interaction through extracellular signal-regulated kinase

Cyclin D1 plays significant roles in cell cycle entry and migration. We have documented that both integrin α3β1 expressions and the number of podocytes were reduced in focal segmental glomerulosclerosis. We wondered whether integrin-extracellular matrix (ECM) interaction was involved in the regulation of cyclin D1 expression, and the possible signaling pathways in mitogen-stimulating podocytes. Cultured podocytes were divided into serum (mitogens/growth factors)-starved and serum-stimulated groups. Reverse transcription polymerase chain reaction was used to detect cyclin D1 mRNA, and Western blot analysis was used to measure protein concentrations of cyclin D1 and extracellular signal-regulated kinase (ERK) activation (p-ERK/ERK). The integrin-ECM interaction was blocked by anti-β1-integrin monoclonal antibody or RGDS (Arg-Gly-Asp-Ser). The MEK inhibitor, U0126, was used to inhibit ERK activation. The results showed that there was little cyclin D1 protein in serum-starved groups, but it was abundant in serum-stimulated groups. Both cyclin D1 mRNA and protein levels were reduced in serum-stimulated podocytes after blocking integrin-ECM interaction. ERK activation in serum-stimulated podocytes was significantly decreased after blocking integrin-ECM interaction. Cyclin D1 mRNA and protein concentrations in serum-stimulated podocytes were reduced after blocking ERK activation by U0126. We demonstrate that integrin-ECM interaction collaborates with mitogens to activate ERK/mitogen-activated protein kinase pathways which are essential for cyclin D1 expression in podocytes.

Abstract 4703: SAHA downregulation of HuR expression is responsible for reduction of cyclin D1 mRNA stability and cell transformation by EGF in Cl41 cells

Abstract Previous studies have reported that anti-cancer activity of suberoylanilide hydroxamic acid (SAHA) is associated with its downregulation of cyclin D1 protein expression at transcription and translation levels. In current studies, we demonstrated that SAHA downregulation of cyclin D1 expression mainly via modulation of cyclin D1 mRNA stability in EGF-induced mouse epidermal Cl41 cell transformation (anchorage-independent colony formation) model. We found that SAHA treatment resulted in dramatic inhibition of EGF-induced cell transformation and cyclin D1 expression. Further studies showed that SAHA downregulation of cyclin D1 expression was only observed in endogenous cyclin D1, but not in reconstitutional cyclin D1 in same cells, excluding the possibility of SAHA regulation of cyclin D1 expression at levels of protein degradation. Moreover, we found that SAHA was able to inhibit EGF-induced cyclin D1 mRNA expression, whereas it did not show any inhibitory effect on cyclin D1 promoter-deriven luciferase reporter activity under same experimental conditions, suggesting that SAHA might downregulate cyclin D1 mRNA stability. This notion was greatly supported by the results showing that co-treatment of cells with SAHA and actinomycin D increased the cyclin D1 mRNA degradation in comparison to that in cells treated with actinomycin D alone. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR protein expression, which is a well-characterized cyclin D1 mRNA-binding protein. Taken together, our results demonstrated that SAHA treatment resulted in downregulation of cyclin D1 expression by inhibition of HuR expression, which led to reduction of cyclin D1 mRNA stability and protein expression, and further in turn caused the inhibition of EGF-induced cell transformation in Cl41 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4703. doi:1538-7445.AM2012-4703

Long non-coding RNA influences radiosensitivity of colorectal carcinoma cell lines by regulating cyclin D1 expression

To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism. Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines(RKO, Lovo) was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR. Lovo(SF2=0.47) was more sensitivity to radiation than RKO(SF2=0.53) according to the outcome of colony formation assay. High-throughput lncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different (38.6%). There was a significant relationship between expression of several lncRNAs and CCND1 gene. Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines(P>0.05), while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines(P<0.05). After exposed to 2 Gy doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines(P<0.05), while P53 and P21 expressions were not different(P>0.05). The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.

Rsf-1 is overexpressed in non-small cell lung cancers and regulates cyclinD1 expression and ERK activity

Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 in primary lung cancer and its biological roles in non-small cell lung cancer (NSCLC) have not been reported. The molecular mechanism of Rsf-1 in cancer aggressiveness remains ambiguous. In the present study, we analyzed the expression pattern of Rsf-1 in NSCLC tissues and found that Rsf-1 was overexpressed at both the mRNA and protein levels. There was a significant association between Rsf-1 overexpression and TNM stage (p=0.0220) and poor differentiation (p=0.0013). Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression resulted in a decrease of colony formation ability and inhibition of cell cycle progression. Rsf-1 knockdown also induced apoptosis in these cell lines. Further analysis showed that Rsf-1 knockdown decreased cyclin D1 expression and phospho-ERK levels. In conclusion, Rsf-1 is overexpressed in NSCLC and contributes to malignant cell growth by cyclin D1 and ERK modulation, which makes Rsf-1 a candidate therapeutic target in lung cancer.

CREPT Accelerates Tumorigenesis by Regulating the Transcription of Cell-Cycle-Related Genes

Tumorigenesis is caused by an uncontrolled cell cycle and the altered expression of many genes. Here, we report a gene CREPT that is preferentially expressed in diverse human tumors. Overexpression of CREPT accelerates tumor growth, whereas depletion of CREPT demonstrates a reversed effect. CREPT regulates cyclin D1 expression by binding to its promoter, enhancing its transcription both in vivo and in vitro, and interacting with RNA polymerase II (RNAPII). Interestingly, CREPT promotes the formation of a chromatin loop and prevents RNAPII from reading through the 3' end termination site of the gene. Our findings reveal a mechanism where CREPT increases cyclin D1 transcription during tumorigenesis, through enhancing the recruitment of RNAPII to the promoter region, possibly, as well as chromatin looping.

Transcriptional Repression of Mad-Max Complex by Human Umbilical Cord Blood Stem Cells Downregulates Extracellular Signal-Regulated Kinase in Glioblastoma

Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr(202)/Tyr(204) retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression.

Clinical Significance of Nucleostemin Expression and Its Correlation With Cyclin D1 Expression in Malignant Ovarian Tumors

To investigate the expression of the nucleostemin (NS) gene in ovarian tumors and its correlation with the expression of cyclin D1. The expression levels of nucleostemin and cyclin D1 proteins were measured by immunohistochemical staining in ovarian tumors and normal ovarian tissues, and the relationship between their levels was analyzed. Nucleostemin gene and cyclin D1 expressions were detected in 28 and 27 specimens of malignant ovarian tumors (93.7% and 90.0%), 4 and 8 specimens of ovarian borderline tumors (40.0% and 80.0%), and in both specimens of benign ovarian tumors, respectively. The expression of NS gene was seen in 5, 13, and 10 specimens of malignant ovarian tumors of high, moderate, and low grade, respectively. The expression of the nucleostemin gene was significantly related to the tumor grade (r = 0.786, P < 0. 05), and a significant relationship between nucleostemin and cyclin D1 expression was found (r = 0.834, P < 0.05). The expression of the nucleostemin gene was not detected in normal ovarian tissue. The expression of the nucleostemin gene in ovarian tumors is closely correlated with origination, progression, and grading of tumors and may serve as a marker in estimating the malignancy of ovarian tumors. The overexpression of cyclin D1 might be correlated with nucleostemin expression. Nucleostemin may have an impact on the passage of cells through the G1/S checkpoint, and thus cell cycle progress, by regulating cyclin D1 expression.

A novel function of EpCAM in oral squamous cell carcinoma cells under anchorage-independent conditions

Epithelial cell adhesion molecule (EpCAM), involved in Ca2+-independent homotypic cell-cell adhesion in epithelial tissues, is overexpressed in several cancer types. Although studies investigating the function of EpCAM in cancers have shown that it plays a role in cell proliferation, invasion and metastasis, the overall function of EpCAM in cancer cells has remained elusive. Here, we report a novel function of EpCAM in multicellular aggregates (MCAs). EpCAM inhibition using RNA interference (RNAi) did not affect cell morphology, proliferation or expression of certain genes, including cyclin D1 in monolayer cultures of the human oral squamous cell carcinoma cell lines HSC-3 or HSC-4. However, in HSC-4 cells cultured as MCAs, suppression of EpCAM significantly reduced the expression levels of cyclin D1. Nuclear localization of the cyclin D1 protein was observed in MCAs of HSC-4 cells but not in MCAs of EpCAM knockdown HSC4 cells, suggesting that EpCAM regulates cyclin D1 expression and localization in HSC-4 cells under anchorage-independent conditions. We propose that targeting EpCAM might result in more efficient therapies under certain conditions of oral squamous cell carcinoma.

Bcr-Abl Mediated-Depletion of Dual-Specificity MAP Kinase Phosphatase, DUSP1, Expression Promotes the Cell Proliferation.

AbstractAbstract 3381 [Background and Aims]MAPK pathways control the cellular processes such as proliferation, differentiation and apoptosis. Abnormal MAPK signaling plays a key role in the development and progression of cancers. The dual-specificity MAPK phosphatases (DUSPs) act as negative regulators of MAPK activity in normal tissues. DUSP1 (dual-specificity MAPK phosphatase-1) is able to inactivate the ERK, p38, and JNK in resulting the cell cycle and apoptosis, and it has been reported to be lost in a variety advanced cancers. However, its function is little known in CML cells. In this study, we have investigated the function of DUSP1 in CML cell proliferation. [Methods]The cells used in this study were human CML cell lines, K562, Meg01 and SHG3 cells. Primary CML cells (ALDHhi cells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of DUSP1 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For cell survival analysis and the levels of p-ERK1/2, p-p38, and p-ATF2 in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with DUSP1 shRNA or cDNA. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with DUSP1 cDNA or shiRNA, or treated with Abl kinase inhibitors. [Results]In CML cell lines, the expressions of DUSP1 mRNA and protein were significantly increased by treatment with Abl kinase inhibitors or transfection with Bcr-Abl shRNA. In CML cells treated with Abl kinase inhibitors or transfection with Bcr-Abl shRNA, CML cell proliferation was inhibited, and the phosphorylation levels of p-ERK1/2, p-p38, and p-ATF2 were markedly decreased compared to the untreated cells.In addition, the overexpression of DUSP1 induced G1 cell cycle arrest indicating down regulation of cyclin D1 expression in CML cells. Moreover, in CML cells transfected with DUSP1 shRNA, the inhibition of CML cell proliferation by the Abl kinase inhibitors were weakened. In CML stem/progenitor cells (ALDHhi cells) obtained from patients with CML, the expression of DUSP1 mRNA was suppressed, and the transfection with Bcr-Abl shRNA or treatment with Abl kinase inhibitors increased the expression of DUSP1 mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion]Our results demonstrated that the expression of DUSP1 was suppressed by Bcr-Abl in CML cells, and the depletion of DUSP1 promotes cell cycle progression through the induction of cyclin D1 expression, and stimulates CML cell proliferation through the continuous phosphorylation of ERK1/2, p38, and ATF2. Moreover, the induction of DUSP1 expression inhibited the proliferation of CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-200b regulates cyclin D1 expression and promotes S-phase entry by targeting RND3 in HeLa cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that inhibit gene expression post-transcriptionally. By regulating their target genes, miRNAs play important roles in tumor generation and development. Recently, the mir-200 family was revealed to inhibit the epithelial-mesenchymal transition, which is viewed as an essential step in early tumor metastasis. Here, we used luciferase assays to demonstrate that mir-200b interacts with predicted target sites in the 3' untranslated region of RND3. In HeLa cells, mir-200b directly reduced the expression of RND3 at the mRNA and protein levels, which thereby promoted expression of the downstream protein cyclin D1 and increased S-phase entry. In conclusion, our study demonstrates a novel role for mir-200b in cell cycle progression and identifies RND3 as a novel mir-200b target.

Cigarette smoke extract promotes proliferation of airway smooth muscle cells in asthmatic rats via regulating cyclin D1 expression

Background Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle. Methods ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE+pcDNA3.1 group and CSE+pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting. Results (1) The percentage of S+G2M phase, absorbance value at 490 nm wavelength (A490) and the expression rate of PCNA protein in CSE group were (31.22±1.17)%, 0.782±0.221, (90.2±7.0)% respectively, which were significantly increased compared with those of control group ((18.36±1.02)%, 0.521±0.109, and (54.1±3.5)%, respectively) (P &lt;0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S+G2M phase, A490 and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P &lt;0.01). (2) The ratios of A490 of cyclin D1 mRNA in CSE group was 0.288±0.034, which was significantly increased compared with that of control group (0.158±0.006) (P &lt;0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A490 of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P &lt;0.01). (3) The ratios of A490 of cyclin D1 protein expression in CSE group was 0.375±0.008, which was significantly increased compared with that of control group (0.268±0.004) (P &lt;0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A490 of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P &lt;0.01). Conclusion CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

Abstract 3074: Neutrophil gelatinase-associated lipocalin inhibits proliferation of human esophageal adenocarcinoma cells both in vitro and in vivo

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of lipocalin family, is a 25-kDa secreted glycoprotein. NGAL modulates multiple biological functions including cell proliferation, invasion, and metastasis. NGAL is upregulated in multiple gastrointestinal cancers including colorectal, pancreatic, and esophageal adenocarcinoma (EAC). Our group recently showed that NGAL over-expression significantly blocked metastasis of pancreatic adenocarcinoma cells [Cancer Research 68(15): 6100-6108, 2008]. However, the biological role of NGAL in EAC is not known. Aim: To examine the biological role of NGAL in human EAC cells both in vitro and in vivo. Methods and Results: We used OE33 (well-differentiated) and Flo-1 (poorly differentiated) EAC cell lines as our model system. We observed that OE33 cells have significantly higher levels of NGAL expression than Flo-1 cells both by qPCR and western blot analyses. To further study the biological role of NGAL in EAC, we created stable NGAL over-expression clones in Flo-1 (Flo1-NGAL) and NGAL under-expression clones in OE33 (OE33-shNGAL) using lentiviral system. We observed that under-expression of NGAL in OE33 significantly increased cell proliferation as determined by clonogenic assays. In contrast, NGAL over-expression in Flo-1 significantly suppressed cell proliferation. We also observed that NGAL under-expression increased cyclin D1 expression, a known cell cycle regulator. This, in part, explained increased EAC cell proliferation secondary to NGAL under-expression. In addition, NGAL over-expression potently suppressed and conversely, NGAL under-expression significantly increased invasion of EAC clones through MatrigelTM in vitro. Furthermore, to confirm our in vitro findings, we observed that OE33-shNGAL clones strikingly increased tumor volume in a subcutaneouos heterotopic xenograft model in nude mice. Conclusion: We demonstrated for the first time that NGAL under-expression increased cell proliferation and invasion in vitro, which could be in part secondary to regulation of cyclin D1 expression. NGAL under-expression also enhanced tumor growth in vivo. Thus, modulation of NGAL can be a novel therapeutic target in progression of EAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3074.

Abstract 965: Mechanistic studies of a dietary combination in cancer prevention: The effects of manganese superoxide dismutase on tumor suppressor gene activation

Abstract Mechanistic Studies of a Dietary Combination in Cancer Prevention: The Effects of MnSOD on Tumor Suppressor Gene Activation Reactive oxygen species can act as signaling transduction molecules modulating various pathways, such as the PI3K/Akt pathway. It is known that this pathway is constitutively activated in tumorigenesis contributing to protein synthesis, cell cycle progression, migration and survival. However, the PI3K/Akt pathway is negatively regulated by the major tumor suppressor protein PTEN. PTEN has dual specificity phosphatase activity regulating Akt phosphorylation as a lipid phosphatase; as well as, regulating cyclin D1 expression as a protein phosphotase. However, these are the roles of cytoplasmic PTEN. Recently, the nuclear roles of PTEN have received significant attention. Nuclear PTEN plays a major role in maintaining cellular homeostasis and stability by contributing to chromosome stability, DNA repair and cell cycle arrest. Previous studies have shown that PTEN nuclear localization is mediated by oxidative stress and may play a role in p53 stabilization. Nevertheless, MnSOD is considered to be a novel tumor suppressor gene. MnSOD has been defined as a nuclear encoded primary antioxidant enzyme localized in the mitochondria. We have shown in previous studies that the dietary combination Protandim reduced tumor incidence and multiplicity via the synergistic induction of superoxide dismutase enzymes. It has been shown that overexpression of MnSOD in in vivo models can suppress tumor formation via modulation of AP-1 binding. Therefore, it is imperative to look at how MnSOD may affect the process of apoptosis and the activation of tumor suppressor genes involved (i.e. PTEN and p53). Using our in vivo models (wildtype, MnSOD knockout, non-transgenic and MnSOD transgenic mice), we investigated how modulation of MnSOD expression may affect PTEN-mediated p53 stabilization, and how this contributes to chemoprevention. In addition, we explored how mutations in p53 may affect the PTEN-p53 interaction and its effects on tumor promotion via a gene transfection approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 965.

Abstract 1718: ErbB-2 acts as coactivator of Stat3 in heregulin-induced breast cancer growth

Abstract We have previously demonstrated that heregulin (HRG), a ligand for ErbB receptors, activates signal transducer and activator of transcription 3 (Stat3) in primary cultures of ErbB-2 overexpressing C4HD cells, from a murine progestin-dependent mammary tumor. ErbB-2 activity is an absolute requirement in the mechanisms of HRG stimulation of Stat3 activity. Recent findings have demonstrated ErbB-2 nuclear migration and its function as a transcription factor. In this work, we studied whether HRG induces ErbB-2 nuclear migration and its interaction with Stat3 in breast cancer cells. We demonstrated that HRG induces ErbB-2 nuclear migration and colocalization with Stat3 in C4HD and human T47D breast cancer cells. Cyclin D1 is a cancer-related gene that contains Stat3 binding sites (GAS sites) but lacks ErbB-2 response elements (HAS sites). By chromatin immunoprecipitation assays (ChIP) and sequential ChIP, we demonstrated that HRG induces in vivo binding of Stat3 and ErbB-2 to the GAS sites of the cyclin D1 promoter. This finding prompted us to evaluate the ability of HRG to regulate cyclin D1 expression. By inhibiting ErbB-2 and Stat3 activation or silencing their expression, we demonstrated that ErbB-2 and Stat3 participate in the mechanism of HRG-induced cyclin D1 protein expression. Next, we explored whether HRG induces cyclin D1 promoter directly via Stat3 binding to its response elements. C4HD and T47D cells transiently transfected with a cyclin D1 promoter luciferase construct showed an enhanced transcriptional activity with HRG treatment. Overexpression of increasing amounts of ErbB-2 wt resulted in a dose-dependent ErbB-2 capacity to enhance Stat3 transcriptional activity induced by HRG. On the other hand, transfection with increasing amounts of an ErbB-2 mutant that is defective in nuclear entry but retains its cell-surface location and functions (ErbB-2ΔNLS) resulted in abrogation of HRG-induced Stat3 activation of cyclin D1 promoter. Finally, we addressed the effect of targeting ErbB-2 in in vivo HRG-dependent growth of C4HD breast tumors. Transfection of C4HD cells with the ErbB-2ΔNLS expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Taken together, these results suggest a new role of ErbB-2 as a coactivator in the mechanism of HRG-induced transcriptional activation of Stat3. We also found nuclear ErbB-2 to be a requisite for HRG stimulation of in vitro and in vivo breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1718.

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm(2), 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3'-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

The anticancer effect of probiotic Bacillus polyfermenticus on human colon cancer cells is mediated through ErbB2 and ErbB3 inhibition

A wealth of data implicates that ErbB receptors have essential roles in tumor development. Probiotic bacteria are known to exert an anticancer activity in animal studies. Bacillus polyfermenticus (B.P.), a probiotic bacterium, has been clinically used for a variety of gastrointestinal disorders in East Asia. Here, we investigated the effect of B.P. on the growth of tumors and its putative mechanism of actions. Conditioned medium of B.P. cultures (B.P. CM) inhibited the growth of human colon cancer cells including HT-29, DLD-1 and Caco-2 cells. Moreover, B.P. CM suppressed colony formation of HT-29 cells cultured on soft agar and reduced carcinogen-induced colony formation of normal colonocytes. Furthermore, data from the mouse xenograft model of human colon cancer cells showed reduced tumor size in B.P. CM-injected mice when compared to E. coli conditioned medium-injected mice. Exposure of B.P. CM to HT-29 cells for 24 hr, 48 hr and 2 weeks reduced ErbB2 and ErbB3 protein expression as well as mRNA levels. Moreover, cyclin D1 expression that is required for ErbB-dependent cell transformation was decreased by B.P. CM. Furthermore, transcription factor E2F-1 that regulates cyclin D1 expression was also decreased by B.P. CM. These results show that B.P. inhibits tumor growth and its anticancer activity occurs, at least in part, through suppressing ErbB2 and ErbB3. Taken together, our study suggests that this probiotic may be clinically used as a prophylactic treatment to prevent colon cancer development.