Overexpression Of CITED2 Research Articles

300-OR: The Transcriptional Coregulator CITED2 Suppresses Expression of IRS-2 in Endothelial Cells and Impairs Vascular Insulin Signaling In Vivo

Endothelial insulin signaling regulates perfusion, leukocyte adhesion, and angiogenesis, and its impairment may contribute to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIF) are known modulators of insulin signaling and we have recently shown that a negative regulator of HIF activity, CBP/p300 interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined the role of CITED2 in endothelial insulin signaling by generating a mouse model with endothelial-specific loss of Cited2. We found that in the aorta and isolated endothelial cells, loss of Cited2 markedly enhanced insulin-stimulated Akt phosphorylation at Ser473 without altering ERK1/2 phosphorylation. Consistent with this observation, Cited2 deletion in endothelial cells increased insulin-stimulated Vegfa expression, endothelial nitric oxide synthase phosphorylation, and cell proliferation. Endothelial cells lacking CITED2 exhibited a 3.6-fold increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, and 66% increase in Irs2 mRNA expression while IRS-1 was unchanged. Similarly, overexpression of CITED2 in endothelial cells decreased IRS-2 levels by 55% without altering IRS-1. Expression of HIF-2α significantly increased transcription of the Irs2 promoter and co-expression of CITED2 abolished this increase. Moreover, ChIP showed that loss of CITED2 increases occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results suggest that loss of CITED2 enhances endothelial insulin signaling through de-repression of HIF-dependent Irs2 expression. The CITED2-p300 interaction can likely be interrupted by a small-molecule drug, and is thus a potential target to improve insulin action on endothelial cells in order to prevent vascular complications of diabetes. Disclosure B. Kunkemoeller: None. K. Chen: None. X. Wang: None. C. Rask-Madsen: None. Funding National Institutes of Health (5T32DK007260)

The transcription factor Vezf1 represses the expression of the antiangiogenic factor Cited2 in endothelial cells

Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1−/− mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1−/− ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1−/− cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1−/− ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.

The role and underlying mechanism of CITED2-mediated insulin signaling pathway in the pathogenesis of diabetic vascular diseases

It is well established that cardiovascular diseases are the leading causes of diabetes-related death. Endothelial dysfunction is widely accepted as the initial and critical factor contributing to diabetic vascular diseases. Insulin resistance may result in vascular endothelial dysfunction, which in turn aggravates diabetic vascular diseases. Via PI3K/Akt signaling pathway, insulin inhibits the function of FoxOs, which, endothelial FoxO1 especially, exerts an important role in atherosclerosis and angiogenesis. In this regard, Wang et al. characterized 10 FoxO1 target genes regulated by insulin in endothelial cells, among which, CITED2, a transcriptional coregulator, was selected to extensively investigate its role and the underlying mechanism of insulin-regulated angiogenesis. CITED2 was significantly increased in vascular endothelial cells in diet-induced mice, ob/ob mice, as well as obese type 2 diabetic patients, all of those models or subjects are accompanied by insulin resistance. In endothelial cells, insulin significantly down-regulated CITED2 expression through insulin receptor-PI3K-Akt-FoxO1 pathway. Inhibition of CITED2 resulted in significant increases in proliferation and tube formation of endothelial cells. Overexpression of CITED2, however, repressed the transactivation of HIF. The study on the mouse model with hind limb ischemia showed that endothelial CITED2 deficiency gave rise to significant increases of expression of endothelin-1, a well-known HIF target gene, induced by ischemia or hypoxia, suggesting that CITED2 inhibited endothelial angiogenesis via suppressing HIF transactivation. In summary, insulin resistance accompanying obesity and type 2 diabetes leads to enhanced CITED2 expression, consequently impairing HIF signaling and proangiogenic capacity in endothelial cells. Inhibition of CITED2 will be a promising novel way to deal with ischemic cardiovascular diseases in diabetic patients. (Chin J Endocrinol Metab, 2017, 33: 637-643) Key words: Endothelial cell; Insulin signaling pathway; FoxO1; CITED2; Angiogenesis

Ectopic expression of CITED2 prior to reprogramming, promotes and homogenises the conversion of somatic cells into induced pluripotent stem cells

Cited2 plays crucial roles in mouse embryonic stem cells self-renewal, the initiation of the somatic reprogramming process into induced pluripotent stem cells (iPSC) and the suppression of cell senescence. Here, we investigated the potential of CITED2 expression in combination with the Oct4, Sox2, Klf4 and c-Myc factors for reprogramming of primary mouse embryonic fibroblasts (MEF) at passage 2 and 4. The ectopic CITED2 expression in primary MEF prior to the onset of the reprogramming process, generated iPSC with less variability in the expression of endogenous pluripotency-related genes. In contrast, part of the MEF reprogrammed without ectopic expression of CITED2 at passage 4 originated partially reprogrammed iPSC or pre-iPSC. However, the overexpression of CITED2 in the pre-iPSC was insufficient to complete the reprogramming process into iPSC. These results indicated that ectopic CITED2 expression at the onset of the reprogramming process in combination with the reprogramming factors promotes a complete and homogeneous conversion of somatic cells into iPSC.

CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells.

SummaryThe transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.

CITED2 Cooperates with Low PU.1 and DNMT3A to Maintain Self-Renewal in Hematopoietic Stem Cells

CITED2 has a conserved role in the maintenance of normal hematopoiesis. We have recently shown that ~70% of acute myeloid leukemia (AML) patients display enhanced CITED2 expression levels. Interfering with CITED2 expression is detrimental for leukemia maintenance in vitro and in vivo, demonstrating that CITED2 is critically important for the survival of leukemic stem cells (LSCs). Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) led to significantly better human engraftment in transplanted NSG mice, consistent with the maintenance of very primitive lin- CD34+ CD38- CD90+ CD45RA- HSCs within the bone marrow 28 weeks after transplantation. Although the CITED2-engrafted mice displayed enlarged spleens, blood development appeared normal, as measured through myeloid, B and T cell staining. This indicates that CITED2 as a single hit is not sufficient to transform human CD34+ cells.CITED2 expression frequently coincides with low expression of the myeloid transcription factor PU.1, suggesting that combined effects, rather than single events are important during AML development. To investigate this, we combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and studied hematopoietic development. CITED2 increased the percentage of immature CD34+ CD38- cells 5-fold, which was not further increased by the additional downregulation of PU.1. However, functional analysis through limiting dilution LTC-iC assays indicated that combining PU.1 down-, with CITED2 upregulation led to a synergistic 8.5-fold increase in LTC-iC frequency, whereas only changing PU.1 or CITED2 induced a respective 1.4 to 3-fold change in HSC frequency. To more stringently assess self-renewal, we cultured transduced cells for 4 weeks on MS5 cells under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently performed CFC assays. Whereas after 4 weeks all groups displayed similar colony numbers, secondary and tertiary replatings demonstrated that self-renewal could only be maintained for more than 10 weeks when CITED2 upregulation was combined with PU.1 downregulation. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+ CD38- HSCs, as replating of CD34+ CD38+ progenitor-derived colonies did not yield new CFCs.In order to investigate the underlying mechanisms, we performed transcriptome analysis on human HSCPs after knockdown of PU.1, overexpression of CITED2 or the combination of both. PU.1Low-CITED2High cells displayed a gene expression pattern different from the PU.1Low or CITED2High only cells, suggesting that the two events have synergistic effects. Some genes, like HLX and SF3B1 have been shown to cause or are mutated in AML, demonstrating that the synergistic changes are related to AML. When comparing the differentially regulated genes in the PU.1Low -CITED2High cells to the gene expression in the Hemaexplorer database, a similar pattern was observed, when compared between AML and normal cells.In order to investigate the effects of the PU.1low CITED2high combination on AML development, we resorted to a PU.1-dependent mouse model of AML development. CITED2 expression in BM cells from PU.1KD/KD mice (in which deletion of an Upstream Regulatory Element leads to an 80% downregulation of PU.1), led to a steady increase of GFP+ cells over time as compared to control cells and demonstrated a dramatic expansion of Gr-1+ Mac-1+ cells, a hallmark of AML in these mice. This suggests that CITED2 contributes to a faster progression towards AML upon lowering of PU.1.To identify if our model corresponds to AMLs with a specific subset of mutations, we clustered publically available AML data (TCGA), based on the gene expression changes in the PU.1Low -CITED2High cells. The majority of AMLs clustered together in 2 groups, in which FLT3, p53 and DNMT3A mutations were most prevalent. FLT3 mutations, through its activation of STAT5, are consistent with high CITED2 expression, whereas p53 mutations are consistent with our data indicating that CITED2 loss regulates HSCs in a p53-dependent manner. The presence of DNMT3A mutations suggests that DNA methylation changes collaborate with high CITED2 and low PU.1 during leukemogenesis. This is currently under investigation.In summary, our data imply that CITED2, low PU.1 and potentially changes in DNA methylation all contribute to maintenance of self-renewal and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

CITED2 Facilitates Apoptosis Induced By Histone Deacetylase Inhibitor SAHA In Human Pediatric Pre-B Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, representing 31% of all tumors, and about 85% of children with ALL have B-cell ALL. Although the survival rate is approaching 90%, ALL remains the main cause of death from disease in children and young adults. The activity of histone deacetylases (HDAC) in childhood ALL is increased compared with that in normal peripheral blood mononuclear cells or bone marrow cells. Treatment of mice engrafted with T or B-ALL cells with HDAC inhibitor (HDACi) increases the acetylation of Histone 3 and Histone 4 and prolongs survival of these mice. <>Vorinostat (Suberoylamilide Hydroxamic Acid, SAHA) was the first HDACi approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Currently, several clinical trials are being conducted to evaluate its effects on other cancers, including ALL. However, some patients are resistant to HDACi therapy, and concerns regarding toxic side effects of HDACi exist due to the roles of HDACs in multiple pathways. Therefore, identification of new therapeutic targets is required which could improve the efficacy of HDACi by reducing the dose of HDACi administered without compromising the treatment benefits but alleviating the side effects of HDACi. <>CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)–rich tail 2 (CITED2) is a cytokine-inducible gene that plays various roles during mouse development and, in particular, is essential for normal hematopoiesis. While the role of CITED2 in the pathogenesis of leukemia is currently unclear, dysregulation of CITED2 has been implicated in various types of leukemia, including ALL, in which downregulation of CITED2 is frequently observed. In this study, we tested the hypothesis that CITED2 may enhance the sensitivity of human pediatric pre-B ALL cells to HDACi SAHA. <>SAHA treatment of NALM-6 and 697 cells (human pediatric pre-B ALL cell lines) significantly induced apoptosis and cell cycle arrest in a dose dependent manner. The protein level of CITED2 was not affected by SAHA treatment. Although overexpression of CITED2 alone only slightly increased apoptosis, it significantly enhanced apoptosis resulted from SAHA treatment in both NALM-6 (15.1% versus 39.2%) and 697 cells (9.4% versus 14.6%) as assessed by annexin V/Propidium Iodide double staining and flow cytometry analysis (Figure 1). Accordingly, compared with control (i.e. NALM-6 cells transduced with GFP), overexpression of CITED2 also greatly reduced mitochondrial membrane potential of NALM-6 cells caused by SAHA treatment. [Display omitted] To explore the potential mechanisms underlying enhanced apoptosis by overexpression of CITED2 in NALM-6 cells treated with SAHA, we determined the levels of pro- and anti- apoptotic proteins by Western blot and real-time quantitative PCR. We found that SAHA treatment increased the levels of pro-apoptotic molecules Bak, Puma, and Noxa, and decreased the levels of anti-apoptotic molecule Bcl-xL and apoptosis inhibitors XIAP and survivin. Importantly, overexpression of CITED2 markedly increased the protein levels of pro-apoptotic molecules Bak and Bim. Furthermore, knockdown of Bim by shRNA significantly attenuated apoptosis in Cited2 overexpressing NALM-6 cells treated with SAHA.Taken together, these results suggest that modulation of the CITED2 activity may confer its cooperative effect with SAHA in pre-B ALL cells and warrant future evaluation of such a combination in inducing apoptosis of primary ALL cells. Disclosures:No relevant conflicts of interest to declare.

Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells.We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells.To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice.Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures:No relevant conflicts of interest to declare.

CITED2 Modulates Differentiation and Proliferation of Normal and Leukemic Hematopoietic Stem and Progenitor Cells Downstream of PU.1

Abstract 922Recently, it was demonstrated that the transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. Little is known regarding the regulation of CITED2, its expression levels in leukemic stem cells (LSCs) and whether CITED2 expression contributes to leukemogenesis. Using microarray data, we found variable CITED2 expression in ∼90% of sorted CD34+ AML cells (n=46). Q-PCR on 12 of these samples indicated that 50% of the leukemic samples displayed higher expression of CITED2 as compared to mobilized peripheral blood (PB) or cord blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSCPs). To verify whether this expression of CITED2 in leukemic cells is functional, we performed RNAinterference on primary CD34+ cells from acute myeloid leukemia (AML) patients (n=9). AML samples with high CITED2 expression were susceptible to lentiviral downregulation of CITED2 as evidenced by the loss of transduced cells from long-term leukemic cultures.To investigate a potential role of CITED2 in leukemogenesis, lentiviral gain-of-function experiments were performed with CB-derived CD34+ HSCPs. These experiments indicate that CITED2 expression increases cell numbers up to 5-fold in long-term culture-initiating cell (LTC-iC) experiments. This cell expansion on MS5-stromal cultures was paralleled by a short-term maintenance of progenitors. Colony-forming-cell (CFC) assays demonstrated a 3.5 fold higher output of CFC colonies compared to control cultures up to 2 weeks of MS-5 co-culture. CITED2-expressing colonies were larger than control colonies, verifying the increased cell numbers in LTC-iC experiments.To further dissect the effects of CITED2, transduced CD34+CD38− HSCs and CD34+CD38+ progenitors were sorted for single-cell liquid cultures and cell-divisions were followed for 6 days. Analysis of 360 single CD34+CD38− HSCs indicated that CITED2 overexpression leads to an enhanced quiescence (cells with no division) and decreased proliferation (cells that divide). However, tracking the divisions of 360 single CD34+CD38+ progenitor cells demonstrated the opposite: Cells overexpressing CITED2 divided more than control cells. These data are consistent with a role for CITED2 in leukemic stem cells (LSCs), where LSCs are thought to be more quiescent than leukemic progenitors.Since leukemias are also characterized by a differentiation block, we subsequently analyzed the differentiation of CB-derived CD34+ HSCPs upon overexpression of CITED2. In LTC-iC experiments, a shift in myelo-monocytic differentiation was observed. Enhanced CITED2 expression led to an increased percentage of CD15-positive cells (64%) at the expense of CD14-positive cells (9%) compared to control cultures (35% and 32% respectively). This bias towards granulocytic differentiation was also observed on May-Grunwald-Giemsa (MGG) stains and was furthermore confirmed in CFC assays. Furthermore, when analyzing erythroid differentiation, a clear reduction in CD71bright GPA+ cells could be observed in CITED2 expressing cells, compared to control cells (2% vs. 12% respectively), which was confirmed by CFC assays and MGG stains.Towards clarifying the regulation of CITED2, we scanned the CITED2 promoter for transcription factor binding sites and identified several PU.1 binding sites. Gene expression comparison between PU.1 and CITED2 in a panel of primary AML samples indicated that, apart from FAB M2 AMLs, PU.1 expression is inversely correlated with CITED2 expression. To functionally investigate this inverse correlation, we performed chromatin immunoprecipitations (ChIP) and demonstrated that PU.1 is indeed able to bind to the PU.1 binding sites in the CITED2 promoter of CB CD34+ cells. Subsequent overexpression of PU.1 in CB CD34+ HSPCs led to a 2-fold reduction in CITED2 expression.Taken together, we propose a model in which PU.1 tightly regulates CITED2 expression during normal myeloid differentiation. In certain AML subsets, this model would predict that low PU.1 expression results in failure to lower CITED2 expression below a certain threshold, which subsequently results in maintenance of LSC quiescence. Furthermore, the enhanced CITED2 levels result in an increased proliferation of downstream leukemic progenitors, where together with low PU.1 levels the normal myeloid differentiation program is perturbed contributing to leukemic development. Disclosures:No relevant conflicts of interest to declare.

Knockdown of CITED2 using short‐hairpin RNA sensitizes cancer cells to cisplatin through stabilization of p53 and enhancement of p53‐dependent apoptosis

CITED2 is a transcriptional modulator which has been implicated in human oncogenesis. In the present study, we examined whether CITED2 is also involved in the resistance of cancer cells to the chemotherapeutic drug cisplatin. We first observed that knockdown of CITED2 using short-hairpin RNA sensitized non-tumorigenic HEK293 cells to cisplatin. Sensitization to cisplatin following knockdown of CITED2 was also observed in cervical carcinoma HeLa cells and in cisplatin-resistant HeLa cells, thereby showing that acquired cisplatin resistance could be reversed by CITED2 knockdown. This sensitization response was dependent on the status of p53 since efficient sensitization was observed in p53-positive hepatocellular carcinoma (HCC) Sk-Hep-1 cells, whereas a negligible response was produced in the two p53-defective cell lines HCC Mahlavu and lung cancer H1299. In contrast, overexpression of CITED2 decreased sensitivity of HEK293 cells to cisplatin, while moderate resistance was produced in HeLa cells. Overexpression of CITED2 also decreased sensitivity to cisplatin in p53-defective H1299 cells when exogenous p53 expression was re-introduced. We observed that knockdown of CITED2-induced CBP/p300-mediated p53 acetylation (Lys373) in HEK293 cells, thereby leading to a decrease of p53 ubiquitination and subsequent accumulation of the p53 protein. Notably, the effects of CITED2 knockdown on p53 accumulation and the increase of p53's target Bax were more pronounced after treatment with cisplatin. Based on these results, we propose that a combination of cisplatin and CITED2 shRNA may represent an effective treatment against p53-sensitive cancer cells.

Abstract 274: Cited2, a novel EGFR-induced coactivator, plays a key role in lung cancer progression

Abstract Aberrant expression and function of epidermal growth factor receptor (EGFR) are reported in most lung cancer cases and are correlated with poor prognosis. However, the role of many downstream effectors associated with abnormal EGFR signaling is not clear in cancer progression. Cited2, a novel transcriptional co-activator was identified to be induced by hyperactive EGFR mutant L858R in lung cancer cells. Overexpression of Cited2 in CL1-0 lung cancer cells allowed cell to survive in serum free condition. Knockdown of Cited2 attenuated cell growth, leading to G1/S cell cycle arrest. Through microarray analysis, we identified that expression of E2F3 and its downstream gene targets were blocked in Cited2-silenced cells. Overexpression of Cited2 enhanced E2F3 expression, supporting that Cited2 regulates E2F3 expression. By chromatin immunoprecipitation assay, we found Cited2 bound to E2F3 promoter. In addition to affecting cell growth, knockdown of Cited2 promoted cell senescence with flat and enlarged cell morphology, and increased senescence-associated ß-galactosidase activity. Through western blot analysis, we found Cited2-silencing induced p53 and p21 expression. Overexpression of Cited2 further enhanced tumor growth in nude mice. Clinical analysis showed that Cited2 was highly expressed in lung cancer but not in normal tissues. These data demonstrate that Cited2 plays a key role in lung cancer progression. 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 274.

Post-transcriptional Control of Cited2 by Transforming Growth Factor β: REGULATION VIA SMADS AND CITED2 CODING REGION

Cited2 is a transcription factor without typical DNA binding domains. Cited2 interacts with cAMP-responsive element-binding protein-binding protein (CBP)/p300, TFAP2, Lhx2, and nuclear receptors, such as peroxisome proliferator-activated receptor and estrogen receptor to function as a transcriptional modulator. Overexpression of Cited2 in Rat1 cells leads to tumor formation in nude mice, suggesting that Cited2 is a transforming gene. Through microarray analysis, Cited2 was found to be down-regulated by transforming growth factor beta1 (TGF-beta) in various cell lines. In this study, we confirmed that both mRNA and protein levels of Cited2 are down-regulated in MDA-MB-231 breast cancer cells. Overexpression of Smad7 or knockdown of Smad4 in MDA-MB-231 cells showed that the Smad pathway is involved in the down-regulation of Cited2. Based on nuclear run-on analysis and Cited2 promoter/reporter assay, Cited2 transcription was not affected by TGF-beta, supporting that down-regulation of Cited2 by TGF-beta is most likely through post-transcriptional regulation. By using transcriptional inhibitors, we demonstrated that the turnover of Cited2 transcripts appears to be accelerated during TGF-beta stimulation. Pharmacologic inhibition of translation with cycloheximide attenuated Cited2 down-regulation by TGF-beta. We examined the expression of recombinant Cited2 gene introduced into MDA-MB-231 cells by stable transfection, and we found that mRNA containing the Cited2 protein-coding region controlled by a heterologous promoter indeed responds to TGF-beta-mediated down-regulation. Study from Cited2 deletion mutants showed that the C-terminal conserved region of Cited2 coding sequence is essential for the down-regulation. This is the first demonstration that TGF-beta-mediated down-regulation of Cited2 is post-transcriptional, through the Smad pathway, and requires the presence of its coding sequence.

Cited2 modulates TGF-β-mediated upregulation of MMP9

Cited (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding domains, has been implicated in control of cell growth and malignant transformation in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-beta signaling. Overexpression of Cited2 enhanced TGF-beta-mediated transcription of a Smad-Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glutathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-beta-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-beta-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-beta-mediated upregulation of MMP9 and TGF-beta-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-beta stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co-activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-beta.