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Cardiac physiological growth is needed for increased demands of heart function after exercise. Prior work suggests that exercise-responsive molecules, including Cbp/P300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain 4 (CITED4), can mediate exercise-induced myocardial physiological growth and promote functional recovery after ischemia–reperfusion injury in adult mice. Moreover, forced expression of CITED4 induces physiological cardiac growth in rodent model. Multiple mouse models of myocardial physiological growth have been established by activating genes such as CITED4, IGF1R and AKT. However, an in vitro model of physiological growth in cardiomyocytes derived from human embryonic stem cells (hESC-CMs) has not yet been developed. To provide clinically relevant models for exploring the molecular mechanism of physiological growth, we generated an inducible hESC cell line with forced CITED4 gene expression and differentiated those hESCs towards cardiomyocytes. The results showed that forced CITED4 expression increased cell size and proliferation in hESC-CMs, and promoted cardiomyocyte proliferation in 3D cardiac microtissues. Activation of protein kinase B (also known as AKT1) signaling was necessary for CITED4-induced proliferation in hESC-CMs and 3D cardiac microtissues, while mTOR signaling mediated both proliferation and physiological hypertrophy induced by CITED4. In an in vitro model mimicking ischemia–reperfusion injury, CITED4 expression inhibited cardiomyocyte apoptosis in hESC-CMs and 3D cardiac microtissues, and this effect was mediated by activation of the mTOR signaling. In conclusion, we successfully generate a physiological growth model in hESC-CMs and 3D cardiac microtissues. Moreover, physiological growth induced by CITED4 is mediated by activation of the mTOR signaling, which is necessary to promote both proliferation and physiological hypertrophy, and to alleviate apoptosis after ischemia–reperfusion injury in hESC-CMs and 3D cardiac microtissues. Graphical abstract Graphical representation of the generation of 2D and 3D myocardial physiological growth model using human embryonic stem cells (hESC). Controlled expression of CITED4 promotes myocardial physiological growth in hESC-derived cardiomyocytes (hESC-CM) and microtissues. AKT activation is necessary for CITED4-induced proliferation of hESC-CMs and 3D cardiac microtissues, while mTOR signaling mediates both proliferation and physiological hypertrophy induced by CITED4. In simulated ischemia–reperfusion injury model, CITED4 expression inhibits cardiomyocyte apoptosis in hESC-CM and microtissues, and this effect is also mediated by activation of mTOR signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by early liver metastasis and high mortality. The tumor microenvironment plays a pivotal role in tumor progression; however, the immune microenvironment's involvement in PDAC liver metastasis remains poorly understood. This study investigates cellular heterogeneity in primary tumor (PT) and liver metastasis (LM) tissues of PDAC using single-nucleus RNA sequencing and spatial transcriptomics. Intra-tumor heterogeneity and cell interactions were elucidated through deconvolution, intercellular signalling, pseudotime analysis, and immune infiltration profiling. The spatial distribution of immune cells was assessed by multiplexed immunofluorescence staining, and prognostic models were developed and validated through immunohistochemistry (IHC). Analyzing the regulatory role of CITED4 in the invasion and metastasis of pancreatic cancer cells through transwell assay and scratch wound healing assay. A total of 62,326 cells were sequenced, with metastatic dissemination cells showing significant upregulation of epithelial-mesenchymal transition (EMT)-related genes during liver metastasis. Spatial transcriptomics revealed the enrichment of metastatic dissemination cells and FOXP3-related T cells at the tumor front in PT tissues. In comparison to LM tissues, the tumor front in PT tissues fosters an immunosuppressive microenvironment through the accumulation of T cells. Interaction analysis identified the SPP1 pathway as a key promoter of this immunosuppressive environment. Furthermore, prognostic models highlighted CITED4 as critical biomarkers in PDAC. Elevated CITED4 expression is correlated with liver metastasis and poor prognosis in patients with PDAC. siRNA-mediated knockdown of CITED4 suppresses the invasion and metastasis of pancreatic cancer cells. In summary, this study revealed that T cell alterations, mediated by metastatic dissemination cells within the immune microenvironment, significantly contribute to PDAC liver metastasis, and that CITED4 enhances the metastatic potential of metastatic dissemination cells.

Background Gemcitabine (GEM) is used as a first-line therapy for patients diagnosed with any stage of pancreatic cancer (PC); however, patient survival is poor because of GEM resistance. Thus, new approaches to overcome GEM resistance in PC are urgently needed. Here, we aimed to establish an in vivo drug-resistant PC model and identify genes involved in GEM resistance. We focused on one of these factors, CITED4, and elucidated its mechanisms of action in GEM resistance in PC. Methods L3.6pl, a GEM-sensitive PC cell line, was orthotopically injected into the pancreas of BALB/c nude mice to establish a GEM-resistant PC animal model. Transcriptomic data from control or GEM-resistant tumor-derived cells were analyzed. GEM resistance was evaluated using cell viability, clonogenicity, and apoptosis assays. An apoptosis array was used to identify genes downstream of CITED4 . A CITED4 knockout-mediated GEM sensitivity assay was performed in an orthotopic xenograft mouse model using PANC-1 cells, which are GEM-resistant cells. Results From the RNA sequencing data of isolated GEM-resistant PC cells and The Cancer Genome Atlas dataset, 15 GEM resistance-related genes were found to be upregulated, including CITED4 , the gene encoding a type of CBP/p300-interacting transactivator implicated in several cancers. CITED4 knockdown in drug-resistant cells reduced cell proliferation and migration but increased apoptosis. To identify the molecular mechanism underlying CITED4-mediated induction of GEM resistance, alterations in Baculoviral IAP Repeat Containing 2 (BIRC2) levels were observed using an apoptosis array. BIRC2 expression was downregulated following CITED4 knockdown in GEM-resistant PC cell lines. Furthermore, chromatin immunoprecipitation and promoter assays showed that BIRC2 was directly regulated by CITED4 . Consistent with the CITED -knockdown experiments, silencing of BIRC2 increased the sensitivity of L3.6pl-GEM-resistant and PANC-1 cell lines to GEM. Furthermore, CITED4 knockout using the CRISPR-Cas9 system in PANC-1 cells increased the sensitivity to GEM in orthotopic mice. Moreover, elevated CITED4 and BIRC2 expression levels were associated with poorer outcomes in human PC clinical samples. Conclusions Collectively, these results indicate that CITED4 regulates GEM resistance via inhibition of apoptosis by upregulating BIRC2 expression in PC cells. Therefore, CITED4 may serve as a valuable diagnostic marker and therapeutic target for GEM-resistant PC.

The CITED proteins function as transcriptional modulators that are essential for vertebrate development. These proteins interact with numerous partners, notably transcription factors and co-activators. The hallmark of the CITED family is their conserved carboxy-terminal domain, which interacts strongly with the CBP/p300 co-activators. The expression of CITED genes is detected early during embryogenesis within embryonic and foetal regions critical for cardiac morphogenesis, among other developmental processes. Notably, CITED2 loss of function is strongly associated with congenital heart malformations in mice and zebrafish embryos, as well as congenital heart disease (CHD) in humans, whereas other CITED family members are not critical for cardiogenesis. Emerging evidence implicates CITED2 and CITED4 in regulating heart physiological adaptations and protective responses to pathological stress. This review provides a detailed analysis of CITED proteins and their interactors, focusing on CITED-target genes relevant for cardiogenesis and heart disease. We also highlight recent findings indicating that CITED2 and CITED4 may be instrumental for the development of novel therapeutic strategies to mitigate CHD and preserve adult cardiac function.

Background CITED4 belongs to the CBP/p300‐interacting transactivator with glutamic acid and aspartic acid‐rich tail (CITED) family which is induced by various cytokines and participates in cytokine‐induced proliferation and differentiation. CITED4 is induced by HB‐EGF in lung cancer cells. However, it is unclear whether and how CITED4 contributes to the invasion and metastasis of lung adenocarcinoma (ADC). Methods CITED4 expression in lung adenocarcinoma and its association with disease‐free survival (DFS) and overall survival were analyzed based on a cohort of 261 patients. The roles of CITED4 were validated via loss‐of‐function and gain‐of‐function experiments. The relationship between CITED4 and CLDN3 was validated by immunohistochemistry, Western blotting, and luciferase reporter assays. The function of the CITED4‐CTNNB1‐CLDN3 complex was fully validated and described. Results CITED4 expression was significantly upregulated in ADC tissues and cells and a predictor for DFS. Downregulation of CITED4 attenuated the proliferation and invasion, whereas CITED4 overexpression enhanced these effects. Overexpression and knockdown of CITED4 resulted in the upregulation and downregulation of CLDN3, respectively. Moreover, CITED4 downregulation suppressed CLDN3‐mediated ADC cell metastasis in vivo. CITED4 was highly expressed and positively correlated with CLDN3. Mechanistically, CITED4 interacted with CTNNB1 and functioned synergistically to enhance CLDN3 transcription. Importantly, CITED4 induced ADC invasion via a CLDN3‐dependent pathway. CITED4 determined the level of CLDN3, which in turn affected the sensitivity of tumors to Clostridium perfringens enterotoxin treatment. Conclusions The CITED4‐CTNNB1‐CLDN3 axis plays a key role in the invasion and metastasis of ADC and provides a novel therapeutic target for lung cancer treatment. CITED4 expression is significantly upregulated in ADC tissues and cells. CITED4 interacted with CTNNB1 and functioned synergistically to enhance CLDN3 transcription. The CITED4‐CTNNB1‐CLDN3 axis plays a key role in the invasion and metastasis of ADC and provides a novel therapeutic target for lung cancer treatment.

Most antidiabetic drugs treat disease symptoms rather than adipose tissue dysfunction as a key pathogenic cause in the metabolic syndrome and type 2 diabetes. Pharmacological targeting of adipose tissue through the nuclear receptor PPARg, as exemplified by glitazone treatments, mediates efficacious insulin sensitization. However, a better understanding of the context‐specific PPARg responses is required for the development of novel approaches with reduced side effects. Here, we identified the transcriptional cofactor Cited4 as a target and mediator of rosiglitazone in human and murine adipocyte progenitor cells, where it promoted specific sets of the rosiglitazone‐dependent transcriptional program. In mice, Cited4 was required for the proper induction of thermogenic expression by Rosi specifically in subcutaneous fat. This phenotype had high penetrance in females only and was not evident in beta‐adrenergically stimulated browning. Intriguingly, this specific defect was associated with reduced capacity for systemic thermogenesis and compromised insulin sensitization upon therapeutic rosiglitazone treatment in female but not male mice. Our findings on Cited4 function reveal novel unexpected aspects of the pharmacological targeting of PPARg. Synopsis The identification of the Cited4 cofactor as a sex‐, tissue‐ and signal‐specific mediator of transcriptional responses to glitazones in adipocyte progenitors reveals unexpected aspects of therapeutic PPARg targeting for insulin sensitization in type 2 diabetes and prediabetes. Cited4 is a glitazone target in human and murine adipocyte progenitors promoting the induction of beige adipocyte differentiation. Cited4 is required for rosiglitazone‐mediated but not beta‐adrenergic induction of thermogenic expression in subcutaneous fat in a sex‐biased manner. Systemic energy expenditure and maximal beta‐adrenergic adipocyte respiration are reduced in Cited4‐deficient female mice under rosiglitazone treatment. Reduced thermogenic expression in subcutaneous fat is associated with compromised insulin sensitization upon therapeutic rosiglitazone treatment specifically in female mice. Graphical Abstract The identification of the Cited4 cofactor as a sex‐, tissue‐ and signal‐specific mediator of transcriptional responses to glitazones in adipocyte progenitors reveals unexpected aspects of therapeutic PPARg targeting for insulin sensitization in type 2 diabetes and prediabetes.

Purpose CITED4 is one member of a family of transcriptional cofactors, several of which are deregulated in a variety of tumors, including colorectal cancer (CRC). We modulated CITED4 expression, in vitro, and analyzed the associated phenotypic and gene expression changes. Methods CITED4-overexpressing and shRNA-mediated knockdown cell lines and control cell lines were established in the CRC cell line SW480. The cells were analyzed for changes in proliferation, apoptosis/cell cycle, migration, invasion, colony formation and adhesion. mRNA expression changes were determined by microarray and pathway analysis, and several deregulated genes were validated by qRT-PCR and Western blotting. Based on results obtained from these studies, the status of the actin cytoskeleton was evaluated by phalloidin/vinculin staining. Results Phenotypically, the CITED4-overexpressing cell line showed only moderate changes in adhesion. Microarray analysis identified several deregulated genes, including several G protein-coupled receptors. Phenotypic analysis of the CITED4 shRNA knockdown cell line demonstrated decreased cell proliferation and G2 cell cycle blockage. Microarray analysis identified many deregulated genes, and pathway analysis discovered genes linked to actin-associated adherens junctions/tight junctions (claudin-4, claudin-7, ezrin, MET, ß-catenin). Phenotypically, no morphological changes of the actin cytoskeleton were seen. Conclusions Upregulation of CITED4 in SW480 resulted in no obvious phenotype. CITED4 shRNA-mediated knockdown led to decreased cellular proliferation and modulation of a large number of genes, including the c-MET tyrosine kinase and several actin-associated adherens junctions/tight junction genes.

Background: Exercise training (ET) has a cardioprotective effect and can induce new cardiomyocyte formation in physiological cardiac remodeling. However, following a large myocardial infarction (MI), the changes in cardiac regeneration and the subsequently different intensities of ET remain unexplored. We investigated the effect of different intensities of ET on the expression of C/EBPβ and CITED4 after an MI. Methods: Adult male Wistar rats were exposed to left coronary artery ligation (LAD). Following an echocardiographic evaluation at four weeks after surgery, MI-operated animals were randomly assigned to either the MI-sedentary (MI-Sed) or the MI-trained groups (low intensity interval training (LIIT), moderate intensity interval training (MIIT), and high intensity interval training (HIIT)). MI-trained animals performed six weeks ET protocols (five days/week). Results: Ten weeks after surgery, HIIT and MIIT animals had significantly higher LV ejection fraction compared to MI-Sed animals. HIIT animals had significantly diminished mRNA levels of C/EBPβ compared to MI-Sed. The mRNA level of CITED4 in HIIT and MIIT were significantly high compared to MI-Sed. Conclusions: The present study demonstrates that cardiac functions are ET intensity-dependent, following a MI. It seems that higher intensities of ET are most effective agents in promoting the expression of C/EBPβ and CITED4. Thus, ET has emerged as an important variable in preclinical and clinical investigations.

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O 2 ) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 ( P  = 0.0029). HIF-1α downstream SLC2A1 was decreased ( P  = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP /p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene ( CITED4 ) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4 -associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2′-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.