CCND1 Overexpression Research Articles

Functional variant rs9344 at 11q13.3 regulates CCND1 expression in multiple myeloma with t(11;14).

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by cytogenetic abnormalities, such as t(11;14)(q13;q32), resulting in CCND1 overexpression. The rs9344 G allele within CCND1 is the most significant susceptibility allele for t(11;14). Sequencing data from 2 independent cohorts, CoMMpass (n = 698) and Mayo Clinic (n = 661), confirm the positive association between the G allele and t(11;14). Among 80% of individuals heterozygous for rs9344 with t(11;14), the t(11;14) event occurs on the G allele, demonstrating a biological preference for the G allele in t(11;14). Within t(11;14), the G allele is associated with higher CCND1 expression and elevated H3K27ac and H3K4me3. CRISPR/Cas9 mediated A to G conversion resulted in increased H3K27ac over CCND1 and elevated CCND1 expression. ENCODE ChIP-seq data supported a PAX5 binding site within the enhancer region covering rs9344, showing preferential binding to the G allele. Overexpression of PAX5 resulted in increased CCND1 expression. These results support the importance of rs9344 G enhancer in increasing CCND1 expression in MM.

Large B-cell lymphomas with CCND1 rearrangement have different immunoglobulin gene breakpoints and genomic profile than mantle cell lymphoma

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL.

Circ-RNF111 Promotes Proliferation of Ovarian Cancer Cell SKOV-3 by Targeting the MiR-556-5p/CCND1 Axis.

The aim of this study was to investigate the role and mechanism of circ-RNF111 in the human ovarian cancer cell line SKOV-3. First, qRT-PCR was used to detect circ-RNF111 and miR-556-5p expression levels in human normal ovarian epithelial cells IOSE80 and human ovarian cancer cells SKOV-3. CCK-8 and colony formation assays were adopted to determine the proliferation rate and cell viability of SKOV-3 cells, respectively. Additionally, in an attempt to reveal the mechanism of circ-RNF111, we predicted the targeting relationship between miR-556-5p and circ-RNF111 as well as miR-556-5p and CCND1 using the circinteractome and TargetScan databases, respectively, and validated their relationship by dual-luciferase reporter assay. The protein expression levels of CCND1 in SKOV-3 cells were detected by Western blot. Based on the above experiments, the expression of circ-RNF111 was found to be up-regulated in SKOV-3, and the knockdown of circ-RNF111 significantly inhibited the proliferation and viability of SKOV-3 cells. Then we confirmed that circ-RNF111 sponged miR-556-5p in SKOV-3 cells to up-regulate CCND1 expression. In addition, simultaneous inhibition of miR-556-5p or overexpression of CCND1 in SKOV-3 cells with knockdown of circ-RNF111 reversed the inhibitory effect of knockdown of circ-RNF111 on the protein expression level of CCND1, cell proliferation rate, and cell viability. In summary, circ-RNF111 promotes the proliferation of SKOV-3 cells by targeting the miR-556-5p/CCND1 axis. Circ-RNF111 may serve as a potential target for ovarian cancer therapy.

FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

FGF19 hormone has pleiotropic metabolic functions, including the modulation of insulin sensitivity, glucose/lipid metabolism and energy homeostasis. On top of its physiological metabolic role, FGF19 has been identified as a potentially targetable oncogenic driver, notably in hepatocellular carcinoma (HCC). Nevertheless, FGF19 remained an attractive candidate for treatment of metabolic disease, prompting the development of analogs uncoupling its metabolic and tumor-promoting activities. Using pre-clinical mice models of somatic mutation driven HCC, we assessed the oncogenicity of FGF19 in combination with frequent HCC tumorigenic alterations: p53 inactivation, CTNNB1 mutation, CCND1 or MYC overexpression. Our data revealed a strong oncogenic cooperation between FGF19 and MYC. Most importantly, we show that this oncogenic synergy is conserved with a FGF19-analog Aldafermin (NGM282), designed to solely mimic the hormone’s metabolic functions. In particular, even a short systemic treatment with recombinant proteins triggered rapid appearance of proliferative foci of MYC-expressing hepatocytes. The fact that FGF19 analog Aldafermin is not fully devoid of the hormone’s oncogenic properties raises concerns in the context of its potential use for patients with damaged, mutation-prone liver.

D-box-binding protein alleviates vascular calcification in rats with chronic kidney disease by activating microRNA-195-5p and downregulating cyclin D1.

Vascular calcification (VC) is a critical complication in chronic kidney disease (CKD), where transcription factors (TFs) and microRNAs (miRs) could potentially play a pivotal role in its pathogenesis and progression. To explore the potential molecular mechanism by which the TF D-box-binding protein (DBP) regulates the miR-195-5p/cyclin D1 (CCND1) axis and its impact on aortic VC in CKD rats, we established a rat model of CKD with VC through a 5/6 nephrectomy procedure. This model was treated with lentivirus overexpressing DBP or CCND1 to analyze their roles in aortic VC. Additionally, an in vitro cell model of VC was induced by high phosphorus. This model underwent transfection with lentivirus overexpressing DBP or miR-195-5p mimic/inhibitor to confirm their regulatory roles in aortic VC in vitro. We assessed the interactions between DBP and miR-195-5p, as well as between miR-195-5p and CCND1. Our results indicated that the expression of DBP and miR-195-5p was reduced, while CCND1 levels were elevated in both the rat and cell models. Overexpression of miR-195-5p inhibited VC in vascular smooth muscle cells (VSMCs). Bioinformatics prediction and dual luciferase assays confirmed that DBP could act as a TF to enhance miR-195-5p expression, with Ccnd1 identified as a downstream target gene of miR-195-5p. Overexpression of DBP inhibited aortic calcification in CKD rats, whereas overexpression of CCND1 produced the opposite effect. In conclusion, the TF DBP can inhibit CCND1 expression through transcriptional activation of miR-195-5p, thereby preventing VC in rats with CKD.

A genetic variant in gene NDUFAF4 confers the risk of non-small cell lung cancer by perturbing hsa-miR-215 binding.

Hsa-microRNA-215 (hsa-miR-215) plays multiple roles in carcinogenesis through regulating its target genes. Genetic variants in hsa-miR-215 target sites thus may affect hsa-miR-215-mRNA interactions, result in altered expression of target genes and even influence cancer susceptibility. This study aimed to investigate the associations of genetic variants which located in the binding sites of hsa-miR-215 with non-small cell lung cancer (NSCLC) susceptibility in the Chinese population and reveal the potential regulatory mechanism of functional variants in NSCLC development. The candidate genetic variants were predicted and screened through bioinformatics analysis based on the degree of complementarity of hsa-miR-215 sequences. The potential effects of genetic variants on the binding ability of hsa-miR-215 and target genes were also predicted. A case-control study with 932 NSCLC patients and 1036 healthy controls was conducted to evaluate the association of candidate genetic variants with NSCLC susceptibility, and an independent case-control study with 552 NSCLC cases and 571 controls were used to further validate the promising associations. Dual luciferase reporter gene assay was applied to explore the regulation of the genetic variants on transcription activity of target gene. Cell phenotyping experiments in vitro and RNA sequencing (RNA-seq) were then carried out to preliminarily explore the potential regulatory mechanisms of the target genes in NSCLC. A total of five candidate genetic variants located in the binding sites of hsa-miR-215 were screened. The two-stage case-control study showed that a variant rs1854268 A > T, which located in the 3' untranslated (3'UTR) region of NDUFAF4 gene, was associated with decreased risk of NSCLC (additive model, odds ratio [OR] = 0.83, 95% confidence interval [CI]: 0.75-0.92, p < 0.001). Functional annotation displayed that rs1854268 A > T might downregulate the expression of NDUFAF4 by enhancing the binding affinity of hsa-miR-215-5p to NDUFAF4 mRNA. Additionally, transient knockdown of the NDUFAF4 could inhibit lung cancer cell migration and promote lung cancer cell apoptosis. Further RNA-seq analysis revealed that the knockdown of NDUFAF4 may affect NSCLC development by downregulating the nuclear factor kappa B (NF-κB) and phosphoinositide 3 kinase-AKT (PI3K-AKT) signaling pathways. Moreover, the overexpression of CCND1 could partially attenuate the effects of NDUFAF4 knock down on lung cancer cell migration and apoptosis, indicating that CCND1 may be involved in the tumor-promoting effects of NDUFAF4 as a downstream molecule of NDUFAF4 gene. In conclusion, the genetic variant rs1854268 (A > T) on NDUFAF4 confers NSCLC susceptibility by altering the binding affinity of hsa-miR-215-5p, thus regulating the expression of NDUFAF4 and subsequently influencing downstream tumor molecules and pathways such as CCND1, NF kappa B, and PI3K-AKT signaling pathways.

#6496 CYCLIN D1 AMELIORATES ACUTE KIDNEY INJURY BY IMPROVING FATTY ACID OXIDATION VIA AMPK PATHWAY

Abstract Background and Aims Acute Kidney Injury (AKI) is a critical condition that is caused by the absence of oxygen during the acute ischemic phase, leading to energy metabolism disturbance and kidney tubular epithelial cell damage. Fatty Acid Oxidation (FAO) is the main source of energy production of renal proximal tubular epithelial cells. Timely promoting FAO, increasing supplies of energy, and promoting cell proliferation are essential to improve kidney injury. However, there is no clinically recognized effective treatment for this. Cyclin D1(CCND1), a member of the cell cycle family, plays a vital role in cell proliferation. Our previous study showed that CCND1 improved AKI by increasing FAO. This study aimed to investigate the role and molecular basis of CCND1 involvement in fatty acid oxidation of AKI. Method CCND1 was evaluated in human kidney proximal tubular epithelial cells (HK-2 cells) and in male C57BL/6J mice (wild type). We investigated the protective role of CCND1 in AKI using a mouse model of ischemia-reperfusion injury, which was treated by transferring CCND1-expressing plasmids in male C57BL/6J mice (wild type) by ultrasound and microbubble-mediated delivery. Eight-week-old male C57BL/6J mice (wild type) were subjected to bilateral renal artery occlusion for 30min followed by 24h of reperfusion. We evaluated FAO, proliferation, and autophagy in vitro and in vivo. In addition, we evaluated the concentrations of blood urea nitrogen and creatinine, evaluated kidney ultrastructure and so on. Results In vivo studies showed that activation of CCND1 prevented AKI-induced lipid accumulation, kidney tubule injury and kidney function declined after ischemia-reperfusion injury. Compared to test control, the treatment significantly (p&amp;lt;0.05) reduced the concentrations of blood urea nitrogen and creatinine. Kidney-specific overexpression of CCND1 increased FAO, promoted proliferation and reduced apoptosis. Mechanistically, CCND1 activated the AMPK pathway, which increased the expression of phosphorylation AMP activated protein kinase (p-AMPK) and upregulated FAO. On the other hand, inhibiting the expression of CCND1 worsened the impairment of FAO and disturbed energy metabolism. Conclusion CCND1 improved FAO and reduced lipid accumulation through the active AMPK pathway in kidney proximal tubular epithelial cells (PTECs). Hence, restoring of the expression of CCND1 may offer a novel therapeutic strategy for treating AKI.

#5349 CYCLIN D1 AMELIORATES ACUTE KIDNEY INJURY BY IMPROVING FATTY ACID OXIDATION VIA AMPK PATHWAY

Abstract Background and Aims Acute kidney injury (AKI) is a life-threatening condition. The absence of oxygen during the acute ischemic phase would disturb energy metabolism and cause kidney tubular epithelial cell damage. Fatty Acid Oxidation (FAO) is the main source of energy production of renal proximal tubular epithelial cells. Timely promoting FAO, increasing supplies of energy, and promoting cell proliferation are essential to improve kidney injury, but there is no clinically recognized effective treatment for this. Cyclin D1(CCND1), a member of the cell cycle family, plays a vital role in cell proliferation. Our previous study found that CCND1 improved AKI accompanied with increased fatty acid oxidation. Therefore, we investigated the role and molecular basis for CCND1 involvement in fatty acid oxidation of AKI. Method CCND1 was evaluated in AKI in human kidney proximal tubular epithelial cells (HK-2 cells) and male C57BL/6J mice (wild type). The protective role of CCND1 in AKI was investigated in a mouse model of ischemia-reperfusion AKI treated by ultrasound-microbubble-mediated kidney-specifically transferring CCND1-expressing plasmids in male C57BL/6J mice (wild type). Eight-week-old male C57BL/6J mice (wild type) were subjected to bilateral renal artery occlusion for 30min followed by 24h of reperfusion. We evaluated FAO, proliferation, and autophagy in vitro and in vivo. In addition, we evaluated the concentrations of blood urea nitrogen and creatinine, evaluated kidney ultrastructure and so on. Results In vivo studies had shown that activation of CCND1 can prevent AKI-induced lipid accumulation, kidney tubule injury and kidney function declined after ischemia-reperfusion injury. Compared to test control, the treatment significantly (p &amp;lt; 0.05) lowered the concentrations of blood urea nitrogen and creatinine. Kidney specific overexpression of CCND1 promoted FAO, promoted proliferation and reduced apoptosis. Mechanistically, CCND1 activated the AMPK pathway, which increased the expression of phosphorylation AMP activated protein kinase (p-AMPK) and upregulated FAO. On the contrary, inhibiting the expression of CCND1 exacerbated impairment of FAO and disturbed energy metabolism. Conclusion Thus, CCND1 improved FAO and reduced lipid accumulation via active AMPK pathway in kidney proximal tubular epithelial cells (PTECs). Hence, reconstruction of the expression of CCND1 may be a novel therapeutic strategy for treating AKI.

#5330 CYCLIN D1 AMELIORATES ISCHEMIA/REPERFUSION-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is a life-threatening condition. The absence of oxygen during the acute ischemic phase would disturb energy metabolism and cause kidney tubular epithelial cell damage. Fatty Acid Oxidation (FAO) is the main source of energy production of renal proximal tubular epithelial cells. Timely promoting FAO, increasing supplies of energy, and promoting cell proliferation are essential to improve kidney injury, but there is no clinically recognized effective treatment for this. Cyclin D1(CCND1), a member of the cell cycle family, plays a vital role in cell proliferation. Our previous study found that CCND1 improved AKI accompanied with increased fatty acid oxidation. Therefore, we investigated the role and molecular basis for CCND1 involvement in fatty acid oxidation of AKI. Method CCND1 was evaluated in AKI in human kidney proximal tubular epithelial cells (HK-2 cells) and male C57BL/6J mice (wild type). The protective role of CCND1 in AKI was investigated in a mouse model of ischemia-reperfusion AKI treated by ultrasound-microbubble-mediated kidney-specifically transferring CCND1-expressing plasmids in male C57BL/6J mice (wild type). Eight-week-old male C57BL/6J mice (wild type) were subjected to bilateral renal artery occlusion for 30min followed by 24h of reperfusion. We evaluated FAO, proliferation, and autophagy in vitro and in vivo. In addition, we evaluated the concentrations of blood urea nitrogen and creatinine, evaluated kidney ultrastructure and so on. Results In vivo studies had shown that activation of CCND1 can prevent AKI-induced lipid accumulation, kidney tubule injury and kidney function declined after ischemia-reperfusion injury. Compared to test control, the treatment significantly (p&amp;lt;0.05) lowered the concentrations of blood urea nitrogen and creatinine. Kidney specific overexpression of CCND1 promoted FAO, promoted proliferation and reduced apoptosis. Mechanistically, CCND1 activated the AMPK pathway, which increased the expression of phosphorylation AMP activated protein kinase (p-AMPK) and upregulated FAO. On the contrary, inhibiting the expression of CCND1 exacerbated impairment of FAO and disturbed energy metabolism. Conclusion Thus, CCND1 improved FAO and reduced lipid accumulation via active AMPK pathway in kidney proximal tubular epithelial cells (PTECs). Hence, reconstruction of the expression of CCND1 may be a novel therapeutic strategy for treating AKI.

Immunohistochemical expression of E-Cadherin and Cyclin D1 in different grades of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy, representing up to 80-90% of all malignant neoplasms of the oral cavity. It results from the multistep accumulation of heterogeneous genetic changes. Important risk factors for OSCC include the use of tobacco or betel quid chewing, alcohol consumption, human papillomavirus and poor nutrition. E-Cadherin as a tumour suppressor gene sets a threshold for Wnt/β-catenin signalling. When expression of E-Cadherin is lost, potentiation of Wnt signalling pathway occurs leading to loss of cell-cell adhesion. The cyclin D1 gene (CCND1) located on chromosome 11q13 encodes a nuclear protein that is the regulatory subunit of Cdk-4 and Cdk-6. Cyclin D1 plays a major role in cell cycle transition from G1 to S phase by contributing to inactivation of the retinoblastoma gene product, and overexpression of CCND1 has been reported in 35-40% cases of OSCC. Considering this, we decided to evaluate and compare the expression of CE-Cadherin and Cyclin D1 in different grades of OSCC. A retrospective study was carried out on 60 formalin-fixed paraffin embedded tissue blocks comprising of 20 cases of well-differentiated OSCC, 20 cases of moderately differentiated OSCC and 20 cases of poorly differentiated OSCC. Diagnosed (using H and E), with oral mucosa taken as control. There was downregulation of E-Cadherin and overexpression of Cyclin D1 in increasing grades of OSCC and the difference was statistically significant. E-Cadherin was localised to membranous and shifted to cytoplasm as the grade worsened. Cyclin D1 was localised to nuclei of cells and the expression was seen more at the peripheral portions of tumour islands depicting the proliferative activity of tumour front. The study revealed a good prognostic role of both E-Cadherin and Cyclin D1 in OSCC. The markers can be used for prognostic as well as therapeutic purposes.

PVT1/miR-16/CCND1 axis regulates gastric cancer progression.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be a vital modulator in tumorigenesis of gastric cancer (GC). However, the detailed regulatory mechanism of PVT1 in GC remains largely unclear. In this work, the expressions of PVT1 and microRNA-16 (miR-16) were detected by quantitative real-time PCR (qRT-PCR) in GC tissues and cell lines. GC cell lines NCI-N87 and MKN45 cell lines were chosen for the following assays. After PVT1 was overexpressed or depleted, CCK-8 and Transwell assays were performed to examine the cell viability and invasive capacity. Cell cycle was analyzed by flow cytometry. The expression of cyclin D1 (CCND1) at mRNA and protein levels was measured by qRT-PCR and western blot. The competitive endogenous RNA molecular mechanism among PVT1, miR-16 and CCND1 was verified by bioinformatics analysis, luciferase-reporter gene assay and RNA immunoprecipitation assay. In the present study, it was revealed that PVT1 expression was remarkably evaluated in GC tissues and cell lines than that in the corresponding control group. PVT1 positively regulated the proliferation, migration and cell cycle progression of GC cells. Besides, miR-16 was identified as a target of PVT1, and CCND1 was identified as a target of miR-16. The depletion of PVT1 promoted the expression of miR-16 and suppressed CCND1 expression. Moreover, either miR-16 inhibitor or CCND1 overexpression plasmid could reverse the promoting effects of PVT1 on the malignant biological behaviors of GC cells. In conclusion, PVT1 promoted CCND1 expression by negatively regulating miR-16 expression to enhance the viability, invasion and cell cycle progression of GC cells.

Protective role of circRNA CCND1 in ulcerative colitis via miR-142-5p/NCOA3 axis

BackgroundIncreasing research indicates that circular RNAs (circRNAs) play critical roles in the development of ulcerative colitis (UC). This study aimed to determine the role of circRNA CCND1 in UC bio-progression, which has been shown to be downregulated in UC tissues.MethodsReverse transcription quantitative polymerase chain reaction was used to determine the levels of circRNA CCND1, miR-142-5p, and nuclear receptor coactivator-3 (NCOA3) in UC tissues and in lipopolysaccharide (LPS)-induced Caco-2 cells. Target sites of circRNA CCND1 and miR-142-5p were predicted using StarBase, and TargetScan to forecast potential linkage points of NCOA3 and miR-142-5p, which were confirmed by a double luciferase reporter-gene assay. Cell Counting Kit 8 and flow cytometry assays were performed to assess Caco-2 cell viability and apoptosis. TNF-α, IL-1β, IL-6, and IL-8 were detected using Enzyme-Linked Immunosorbent Assay kits.ResultsCircRNA CCND1 was downregulated in UC clinical samples and LPS-induced Caco-2 cells. In addition, circRNA CCND1 overexpression suppressed LPS-induced apoptosis and inflammatory responses in Caco-2 cells. Dual-luciferase reporter-gene assays showed that miR-142-5p could be linked to circRNA CCND1. Moreover, miR-142-5p was found to be highly expressed in UC, and its silencing inhibited LPS-stimulated Caco-2 cell apoptosis and inflammatory responses. Importantly, NCOA3 was found downstream of miR-142-5p. Overexpression of miR-142-5p reversed the inhibitory effect of circRNA CCND1-plasmid on LPS-stimulated Caco-2 cells, and the effects of miR-142-5p inhibitor were reversed by si-NCOA3.ConclusionCircRNA CCND1 is involved in UC development by dampening miR-142-5p function, and may represent a novel approach for treating UC patients.

EVI1 Promotes Immune-Evasive Microenvironment Via Cyclin D1 in Acute Myeloid Leukemia

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1high (EVI1+) AML cells do not show clear resistance to cytotoxic agents in vitro. Since EVI1 is essential for normal hematopoietic stem cells (HSCs), we performed chromatin-immunoprecipitation (ChIP)- and RNA-sequencing of murine EVI1+ AML cells and normal hematopoietic cells to identify factors involved explicitly in AML, and thereby to elucidate molecular cues to refractory EVI1+ AML. We firstly employed a murine EVI1-transformed AML (EVI1-AML) model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we next performed ChIP coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody and transcriptome analysis by RNA-seq. We further generated another murine EVI1+ AML model by retrovirally transducing the KMT2A-MLLT1 fusion gene into Lineage- Sca-1+ c-kit+ cells from EVI1-GFP knock-in mice, where Evi1-IRES-GFP allele was inserted into the Evi1 locus to enable real-time monitoring of Evi1 expression by GFP, followed by transplantation into lethally irradiated syngeneic mice. Intriguingly, GFPhigh L-GMPs from KMT2A-MLLT1 AML mice showed distinct expression profiles from GFPlow L-GMPs, including high NF-kB activity and low stem cell activity, along with the lower frequency of leukemia-initiating cells in GFPhigh fraction. Consistently, an exogenous expression of EVI1 in KMT2A-MLLT1 AML cells without Evi1 expression decreased colony-forming and leukemia-initiating activity, suggesting a differential role of EVI1 in AML from normal HSCs, where EVI1 is essential in maintaining stem cell function. Combined with functional screening by cell proliferation-related short hairpin RNAs (shRNAs), we identified that cyclin D1 (CCND1) was a downstream target, commonly implied in the above analyses, and a potential therapeutic vulnerability of EVI1-AML. EVI1 bound to the region 3.8 kb upstream of the transcription start site of Ccnd1, through which EVI1 demonstrated strong transcriptional activity in a luciferase reporter assay. Genetic silencing of Ccnd1 and pharmacologic inhibition of Cyclin D1 in EVI1- and EVI1+ KMT2A-MLLT1 AML cells negatively affected proliferation and colony-forming activity in vitro. Moreover, in vivo knockdown of Ccnd1 strikingly impaired the leukemogenic capacity of murine EVI1-AML models. It was surprising that transcriptomic analysis of EVI1-AML cells with Ccnd1 silencing showed the downregulation of genes involved in chemokine production and response to interferon instead of cell cycle-related genes. The expression of Cd274 (PD-L1) as well as Stat1, the primary signaling mediator for type I and II IFN, was decreased in EVI1-AML cells with Ccnd1 knockdown. EVI1 directly bound to the promoter region of Stat1 and activated its transcription in line with a previous report, and indirectly up-regulated STAT1 expression via Cyclin D1. Accordingly, infiltrating CD4 and CD8 T cells from EVI1-shCcnd1 AML mice demonstrated decreased exhaustion markers, including LAG3, PDCD1, and TIGIT. Silencing of Stat1 and Ifngr, the receptor for IFN-γ, impaired the leukemogenic capacity of EVI1-AML in vivo, with a decrease in T and NK cells with exhaustion phenotypes, while Ifnar, the receptor for IFN-α, was not involved in this process. Genetic inhibition of the IFN-γ-STAT1 axis in non-EVI1+ AML models did not affect AML development, nor the frequency of immune cells expressing exhaustion-related molecules, suggesting this axis is specifically important in EVI1+ AML. By using human AML datasets, overexpression of EVI1 and CCND1 were associated with IFN-γ signature, including up-regulation of STAT1 and CD274, and elevated expression of chemokines, with increased exhaustion molecules in T cells. These data collectively suggest that cyclin D1 is implicated in the development of EVI1-AML through the formation of IFN-γ signature and exhausted T cell phenotypes, which could be potentially targeted.

53. Clinical whole exome/whole transcriptome (WES/WTS) analysis detects copy number and structural rearrangements important

<h3>Background</h3> Chromosomal copy number alterations (CNAs) and structural rearrangements (SR) are a key part of risk stratification and management of patients with MM. We developed a clinical paired tumor/normal WES/WTS assay that detects CNAs at one-copy difference from focal to whole genome, and gene fusion at 20% and 10% limit of detection, respectively. <h3>Patients and Methods</h3> We performed WES/WTS analysis utilizing DNA/RNA extracted from CD138+ enriched cells and buccal swab from 92 MM patients. Plasma cell content was not available for most cases. We compared the detection of CNAs, and SR with other techniques currently in use including FISH, arrays and molecular analysis. <h3>Results</h3> Molecular alterations (MA) were present in 82/92(89.1%) cases, whereas 65/92(69.5%) cases had CNAs. In cases with CNAs (indicating sufficient tumor content), deletions of (1p32), (1p12), or both were detected in 7(10.8%), 12(18.5%), and 5(7.7%) cases, respectively. Gain of (1q22), and hyperdiploidy were detected in 19(29.2%) and 32(49.2%), respectively. Deletions of 13q, 14q, 16q, and 17p(TP53) were detected in 32(49.2%), 14(21.6%), 16(24.6%), and 13(20%) cases, respectively. In cases with MA, 10/82(10.8%), and 3(3.6%) cases had TP53 mono and bi-allelic inactivation, respectively. Translocations (4;14)(p16;q32)(IGH::NSD2/FGFR3) and t(14;16)(q32;q23)(IGH::MAF) were detected in 11(11.9%), and 3(3.7%) cases with MA, respectively. Overexpression of CCND1 (research use only) was observed in 8/82(9.7%) cases. <h3>Discussion</h3> Our WES/WTS detected abnormalities in this series similarly to what has been reported by techniques currently needed in combination such as FISH, arrays or molecular analysis. By incorporating RNA expression analysis, WES/WTS may reliably detect all clinically-relevant alterations in MM.

Whole Transcriptome Sequencing Reveals Cancer-Related, Prognostically Significant Transcripts and Tumor-Infiltrating Immunocytes in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) subtype characterized by overexpression of CCND1 and SOX11 genes. It is generally associated with clinically poor outcomes despite recent improvements in therapeutic approaches. The genes associated with the development and prognosis of MCL are still largely unknown. Through whole transcriptome sequencing (WTS), we identified mRNAs, lncRNAs, and alternative transcripts differentially expressed in MCL cases compared with reactive tonsil B-cell subsets. CCND1, VCAM1, and VWF mRNAs, as well as MIR100HG and ROR1-AS1 lncRNAs, were among the top 10 most significantly overexpressed, oncogenesis-related transcripts. Survival analyses with each of the top upregulated transcripts showed that MCL cases with high expression of VWF mRNA and low expression of FTX lncRNA were associated with poor overall survival. Similarly, high expression of MSTRG.153013.3, an overexpressed alternative transcript, was associated with shortened MCL survival. Known tumor suppressor candidates (e.g., PI3KIP1, UBXN) were significantly downregulated in MCL cases. Top differentially expressed protein-coding genes were enriched in signaling pathways related to invasion and metastasis. Survival analyses based on the abundance of tumor-infiltrating immunocytes estimated with CIBERSORTx showed that high ratios of CD8+ T-cells or resting NK cells and low ratios of eosinophils are associated with poor overall survival in diagnostic MCL cases. Integrative analysis of tumor-infiltrating CD8+ T-cell abundance and overexpressed oncogene candidates showed that MCL cases with high ratio CD8+ T-cells and low expression of FTX or PCA3 can potentially predict high-risk MCL patients. WTS results were cross-validated with qRT-PCR of selected transcripts as well as linear correlation analyses. In conclusion, expression levels of oncogenesis-associated transcripts and/or the ratios of microenvironmental immunocytes in MCL tumors may be used to improve prognostication, thereby leading to better patient management and outcomes.

Regulation of Cyclin D1 Degradation by Ubiquitin-Specific Protease 27X Is Critical for Cancer Cell Proliferation and Tumor Growth.

As a deubiquitinating enzyme, USP27X is a druggable target. Our study illuminates new avenues for therapeutic intervention in CCND1-driven cancers.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

Long noncoding RNA ANRIL up-regulates CCND1 via sponging miR-98-5p to promote TGF-β1-induced human airway smooth muscle cell proliferation, migration, and extracellular matrix deposition.

Excessive proliferation and migration of airway smooth muscle cell (ASMC) contribute to asthma pathogenesis. Long noncoding RNAs (lncRNAs) are reported to take part in asthma pathogenesis. This study is targeted at deciphering the role of the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) in ASMC proliferation, migration and extracellular matrix (ECM) deposition. qRT-PCR was performed to determine ANRIL, miR-98-5p, and cyclin D1 (CCND1) mRNA expression levels in transforming growth factor-β1 (TGF-β1)-treated ASMCs. CCK-8 and Transwell assays were employed to examine ASMC proliferation and migration, respectively. Dual-luciferase reporter gene assay and RNA immunoprecipitation assay were carried out for analyzing the targeted relationship of miR-98-5p with ANRIL or CCND1 mRNA 3'-UTR. The levels of CCND1 and ECM proteins (such as fibronectin, COL3A1, and COL1A2) in ASMCs were detected through Western blot. In this work, we found that ANRIL and CCND1 were up-regulated in TGF-β1-treated ASMCs, whereas miR-98-5p was down-regulated. ANRIL overexpression facilitated the proliferation, ECM deposition and migration of TGF-β1-induced ASMCs, while knocking down ANRIL had the opposite effect. Furthermore, ANRIL targeted miR-98-5p directly, and CCND1 was miR-98-5p's downstream target. ANRIL indirectly increased CCND1 expression in ASMCs via competitively binding to miR-98-5p. MiR-98-5p inhibition or CCND1 overexpression counteracted the inhibiting effect that ANRIL knockdown had on TGF-β1-stimulated ASMC proliferation, migration and ECM deposition. In conclusion, ANRIL indirectly up-regulates CCND1 expression by targeting miR-98-5p to promote ASMC proliferation, migration and ECM deposition, thus facilitating the pathogenesis of asthma.

Circular RNA RHOT1 Regulates miR-142-5p/CCND1 to Participate in Chondrocyte Autophagy and Proliferation in Osteoarthritis

Background Osteoarthritis (OA) serves as one of the most prevalent types of joint disorders and is a leading cause of symptoms of stiffness, swelling, and arthralgia. Circular RNAs (circRNAs) have been reported to participate in various cellular processes by competing with microRNAs. Meanwhile, cyclinD1 (CCND1) is a key cell cycle regulatory protein and plays a crucial role in OA progression. Nevertheless, the function of circRHOT1 in the modulation of OA progression is still obscure. Here, we explored the effect of circRHOT1 on autophagy and extracellular matrix (ECM) in OA. Methods The expression of circRHOT1 and autophagy markers was detected in OA samples. The effect of circRHOT1 on OA was analyzed in the OA rat model. The function of circRHOT1 in the regulation of OA was assessed by CCK-8, colony formation, flow cytometry analysis, quantitative real-time PCR, Western blot analysis, and luciferase reporter gene assay in chondrocytes. Results We observed that autophagy markers, including LC3 and beclin1, were repressed in clinical OA samples. The expression of circRHOT1 and CCND1 was induced but the miR-142-5p expression was reduced in clinical OA samples. The miR-142-5p expression was negatively correlated with circRHOT1 and CCND1, and the circRHOT1 expression was positively associated with CCND1 in clinical OA samples. Meanwhile, the apoptosis was induced in OA rats but the depletion of circRHOT1 could block the phenotype in the rats. The articular cartilage degeneration was promoted in OA rats, while the knockdown of circRHOT1 repressed the degeneration. The serum levels of CTX-II and COMP were increased in OA rats, and the silencing of circRHOT1 downregulated levels of these proteins. In addition, the expression of collagen II and aggrecan was promoted by the depletion of circRHOT1 in the OA rats. Significantly, the expression of LC3 and beclin1 was suppressed in OA rats, in which the knockdown of circRHOT1 could reverse the effect. Moreover, the depletion of circRHOT1 repressed the cell viability and proliferation but induced apoptosis of chondrocytes. The expression levels of LC3, beclin1, collagen II, and aggrecan were induced by circRHOT1 knockdown. Mechanically, circRHOT1 was able to enhance the CCND1 expression by sponging miR-142-5p in chondrocytes. The overexpression of CCND1 or the inhibition of miR-142-5p reversed circRHOT1 depletion-mediated chondrocyte phenotypes. Conclusions Circular RNA RHOT1 enhances the CCND1 expression by sponging miR-142-5p to inhibit chondrocyte autophagy and promote chondrocyte proliferation in osteoarthritis. Our findings provided a promising therapeutic target for OA.

Cyclin D1 mediated by the nuclear translocation of nuclear factor kappa B exerts an oncogenic role in lung cancer

ABSTRACT The relevance of cyclin D1 (CCND1) has been implicated in lung cancer progression. Nevertheless, the mechanism by which CCND1 supports lung cancer development is yet to be expounded. Here, we established that CCND1 is overexpressed in clinical lung cancer specimens and various lung cancer cells. Importantly, CCND1 overexpression enhanced lung cancer cell proliferation, invasion and migration, and arrested the cell cycle at the S phase. In vivo, overexpression of CCND1 promoted lung cancer growth and metastasis. The nuclear translocation of nuclear factor kappa B (NF-κB) promoted p65 protein expression and CCND1 transcription. Meanwhile, PI3K/AKT pathway activity was significantly reduced when NF-κB nuclear translocation was decreased. PI3K/AKT pathway activity was significantly elevated upon CCND1 overexpression. Inhibition of PI3K/AKT pathway activity or suppression of NF-κB translocation in cells with high CCND1 expression was found to significantly reduce the activity of lung cancer cells in vitro and in vivo. Our data revealed that NF-κB/CCND1/PI3K/AKT axis could act as a prospective diagnostic biomarker and a therapeutic option for lung cancer.