Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns.

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation.

High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.

Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs.

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.

Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase.

The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer.

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells.

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

Histone demethylase PHF8 promotes prostate cancer metastasis via the E2F1-SNAI1 axis.

Metastasis is the primary culprit behind cancer-related fatalities in multiple cancer types, including prostate cancer. Despite great advances, the precise mechanisms underlying prostate cancer metastasis are far from complete. By using a transgenic mouse prostate cancer model (TRAMP) with and without Phf8 knockout, we have identified a crucial role of PHF8 in prostate cancer metastasis. By complexing with E2F1, PHF8 transcriptionally upregulates SNAI1 in a demethylation-dependent manner. The upregulated SNAI1 subsequently enhances epithelial-to-mesenchymal transition (EMT) and metastasis. Given the role of the abnormally activated PHF8/E2F1-SNAI1 axis in prostate cancer metastasis and poor prognosis, the levels of PHF8 or the activity of this axis could serve as biomarkers for prostate cancer metastasis. Moreover, targeting this axis could become a potential therapeutic strategy for prostate cancer treatment. © 2024 The Pathological Society of Great Britain and Ireland.

Transcriptional mechanism of E2F1/TFAP2C/NRF1 in regulating KANK2 gene in nephrotic syndrome.

The mortality rate linked with nephrotic syndrome (NS) is quite high. The renal tubular injury influences the response of NS patients to steroid treatment. KN motif and ankyrin repeat domains 2 (KANK2) regulates actin polymerization, which is required for renal tubular cells to maintain their function. In this study, we found that the levels of KANK2 in patients with NS were considerably lower than those in healthy controls, especially in NS patients with acute kidney injury (AKI). To get a deeper understanding of the KANK2 transcriptional control mechanism, the core promoter region of the KANK2 gene was identified. KANK2 was further found to be positively regulated by E2F Transcription Factor 1 (E2F1), Transcription Factor AP-2 Gamma (TFAP2C), and Nuclear Respiratory Factor 1 (NRF1), both at mRNA and protein levels. Knocking down E2F1, TFAP2C, or NRF1 deformed the cytoskeleton of renal tubular cells and reduced F-actin content. EMSA and ChIP assays confirmed that all three transcription factors could bind to the upstream promoter transcription site of KANK2 to transactivate KANK2 in renal tubular epithelial cells. Our study suggests that E2F1, TFAP2C, and NRF1 play essential roles in regulating the KANK2 transcription, therefore shedding fresh light on the development of putative therapeutic options for the treatment of NS patients.

Biphasic regulation of miR-17∼92 transcription during hypoxia: roles of HIF1 and p53 hyperphosphorylation at ser15.

We have reported previously that during hypoxia exposure, the expression of mature miR-17∼92 was first upregulated and then downregulated in pulmonary artery smooth muscle cells (PASMC) and in mouse lungs in vitro and in vivo. Here, we investigated the mechanisms regulating this biphasic expression of miR-17∼92 in PASMC in hypoxia. We measured the level of primary miR-17∼92 in PASMC during hypoxia exposure and found that short-term hypoxia exposure (3% O2, 6 h) induced the level of primary miR-17∼92, whereas long-term hypoxia exposure (3% O2, 24 h) decreased its level, suggesting a biphasic regulation of miR-17∼92 expression at the transcriptional level. We found that short-term hypoxia-induced upregulation of miR-17∼92 was hypoxia-inducible factor 1α (HIF1α) and E2F1 dependent. Two HIF1α binding sites on miR-17∼92 promoter were identified. We also found that long-term hypoxia-induced suppression of miR-17∼92 expression could be restored by silencing of p53. Mutation of the p53-binding sites in the miR-17∼92 promoter increased miR-17∼92 promoter activity in both normoxia and hypoxia. Our findings suggest that the biphasic transcriptional regulation of miR-17∼92 during hypoxia is controlled by HIF1/E2F1 and p53 in PASMC: during short-term hypoxia exposure, stabilization of HIF1 and induction of E2F1 induce the transcription of miR-17∼92, whereas during long-term hypoxia exposure, hyperphosphorylation of p53 suppresses the expression of miR-17∼92.NEW & NOTEWORTHY We showed that the biphasic transcriptional regulation of miR-17∼92 during hypoxia is controlled by two distinct mechanisms: during short-term hypoxia exposure, induction of HIF1 and E2F1 upregulates miR-17∼92. Longer hypoxia exposure induces hyperphosphorylation of p53 at ser15, which leads to its binding to miR-17∼92 promoter and inhibition of its expression. Our findings provide novel insights into the spatiotemporal regulation of miR-17∼92 that may play a role in the development of human lung diseases including pulmonary hypertension (PH).

SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

Bleomycin induces senescence and repression of DNA repair via downregulation of Rad51.

BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

CDCA5 accelerates progression of breast cancer by promoting the binding of E2F1 and FOXM1.

BACKGROUND: Breast cancer is one of the most common malignant tumors in women. Cell division cycle associated 5 (CDCA5), a master regulator of sister chromatid cohesion, was reported to be upregulated in several types of cancer. Here, the function and regulation mechanism of CDCA5 in breast cancer were explored. METHODS: CDCA5 expression was identified through immunohistochemistry staining in breast cancer specimens. The correlation between CDCA5 expression with clinicopathological features and prognosis of breast cancer patients was analyzed using a tissue microarray. CDCA5 function in breast cancer was explored in CDCA5-overexpressed/knockdown cells and mice models. Co-IP, ChIP and dual-luciferase reporter assay assays were performed to clarify underlying molecular mechanisms. RESULTS: We found that CDCA5 was expressed at a higher level in breast cancer tissues and cell lines, and overexpression of CDCA5 was significantly associated with poor prognosis of patients with breast cancer. Moreover, CDCA5 knockdown significantly suppressed the proliferation and migration, while promoted apoptosis in vitro. Mechanistically, we revealed that CDCA5 played an important role in promoting the binding of E2F transcription factor 1 (E2F1) to the forkhead box M1 (FOXM1) promoter. Furthermore, the data of in vitro and in vivo revealed that depletion of FOXM1 alleviated the effect of CDCA5 overexpression on breast cancer. Additionally, we revealed that the Wnt/ß-catenin signaling pathway was required for CDCA5 induced progression of breast cancer. CONCLUSIONS: We suggested that CDCA5 promoted progression of breast cancer via CDCA5/FOXM1/Wnt axis, CDCA5 might serve as a novel therapeutic target for breast cancer treatment.

p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Aspongopus chinensis ach-miR-276a-3p induces breast cancer cell cycle arrest by targeting APPL2 to regulate the CDK2-Rb-E2F1 signaling pathway.

Breast cancer, the most common cancer, presents a significant challenge to the health and longevity of women. Aspongopus chinensis Dallas is an insect with known anti-breast cancer properties. However, the anti-breast cancer effects and underlying mechanisms have not been elucidated. Exogenous microRNAs (miRNAs), which are derived from plants and animals, have been revealed to have notable capacities for controlling the proliferation of cancerous cells. To elucidate the inhibitory effects of miRNAs derived from A. chinensis and the regulatory mechanism involved in the growth of breast cancer cells, miRNA sequencing was initially employed to screen for miRNAs both in A. chinensis hemolymph and decoction and in mouse serum and tumor tissue after decoction gavage. Subsequently, the experiments were performed to assess the suppressive effect of ach-miR-276a-3p, the miRNA screened out from a previous study, on the proliferation of MDA-MB-231 and MDA-MB-468 breast cancer cell lines in vitro and in vivo. Finally, the regulatory mechanism of ach-miR-276a-3p in MDA-MB-231 and MDA-MB-468 breast cancer cells was elucidated. The results demonstrated that ach-miR-276a-3p notably inhibited breast cancer cell proliferation, migration, colony formation, and invasion and induced cell cycle arrest at the G0/G1 phase. Moreover, the ach-miR-276a-3p mimics significantly reduced the tumor volume and weight in xenograft tumor mice. Furthermore, ach-miR-276a-3p could induce cell cycle arrest by targeting APPL2 and regulating the CDK2-Rb-E2F1 signaling pathway. In summary, ach-miR-276a-3p, derived from A. chinensis, has anti-breast cancer activity by targeting APPL2 and regulating the CDK2-Rb-E2F1 signaling pathway and can serve as a promising candidate anticancer agent.

Pulsed Electromagnetic Fields Combined With Adipose-Derived Stem Cells Protect Ischemic Myocardium by Regulating miR-20a-5p/E2F1/p73 Signaling.

Myocardial infarction (MI) is a serious threat to human health. Although monotherapy with pulsed electromagnetic fields (PEMFs) or adipose-derived stem cells (ADSCs) has been reported to have positive effect on the treatment of MI, a satisfactory outcome has not yet been achieved. In recent years, combination therapy has attracted widespread interest. Herein, we explored the synergistic therapeutic effect of combination therapy with PEMFs and ADSCs on MI and found that the combination of PEMFs and ADSCs effectively reduced infarct size, inhibited cardiomyocyte apoptosis and protected the cardiac function in mice with MI. In addition, bioinformatics analysis and RT-qPCR showed that the combination therapy could affect apoptosis by regulating the expression of miR-20a-5p. A dual-luciferase reporter gene assay also confirmed that the miR-20a-5p could target E2F transcription factor 1 (E2F1) and inhibit cardiomyocyte apoptosis by regulating the E2F1/p73 signaling pathway. Therefore, our study systematically demonstrated the effectiveness of combination therapy on the inhibition of cardiomyocyte apoptosis by regulating the miR-20a-5p/E2F1/p73 signaling pathway in mice with MI. Thus, our study underscored the effectiveness of the combination of PEMFs and ADSCs and identified miR-20a-5p as a promising therapeutic target for the treatment of MI in the future.

The association of E2F1 and E2F2 single nucleotide polymorphisms with laryngeal squamous cell carcinoma pathomorphological features.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common types of cancer in the upper respiratory tract. It is well-known that it has a high mortality rate and poor prognosis in advanced stages. There are well-known risk factors for LSCC, though new specific and prognostic blood-based markers for LSCC development and prognosis are essential. The current study aimed to evaluate the impact of four different single nucleotide polymorphisms (SNPs), E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028), on LSCC development, morphological features, and patient 5-year survival rate. METHODS: A total of 200 LSCC patients and 200 controls were included in this study; both groups were matched by age and sex. In the present study, we analyzed four single nucleotide polymorphisms (SNPs) in the genes E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028) and evaluated their associations with the risk of LSCC development, its clinical and morphological manifestation, and patients 5-year survival rate. Genotyping was carried out using RT-PCR. RESULTS: None of the analyzed SNPs showed a direct association with LSCC development. E2F2 rs2075993 G allele carriers (OR = 4.589, 95% CI 1.050-20.051, p = 0.043) and rs3820028 A allele carriers (OR = 4.750, 95% CI 1.088-20.736, p = 0.038) had a statistically significantly higher risk for poor differentiated or undifferentiated LSCC than non-carriers. E2F1 rs3213180 GC heterozygotes were found to have a 3.7-fold increased risk for lymph node involvement (OR = 3.710, 95% CI 1.452-9.479, p = 0.006). There was no statistically significant association between investigated SNPs and patient 5-year survival rate. CONCLUSIONS: The present study indicates that E2F2 rs2075993 and rs3820028 impact LSCC differentiation, whereas E2F1 rs3213180 - on lymph node involvement.

New link between RNH1 and E2F1: regulates the development of lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a non-small cell carcinoma. Ribonuclease/angiogenin inhibitor 1 (RNH1) exerts multiple roles in virous cancers. E2F1 is a critical transcription factor involved in the LUAD development. Here, we analyze the expression of RNH1 in LUAD patients, investigate the biological function of RNH1 in LUAD, and demonstrate its potential mechanisms through E2F1 in LUAD. METHODS: In the present study, we presented the expression of RNH1 in LUAD based on the database and confirmed it by western blot detection of RNH1 in human LUAD tissues. Lentiviral infection was constructed to silence or overexpress RNH1 in NCI-H1395 and NCI-H1437 cells. We assess the role of RNH1 on proliferation in LUAD cells by MTT assay, colony formation assays, and cell cycle detection. Hoechst staining and flow cytometry were used to evaluate the effects of RNH1 on apoptosis of LUAD cells. The function of RNH1 in invasion and migration was investigated by Transwell assay. Dual luciferase assay, ChIP detection, and pull-down assay were conducted to explore the association of E2F1 in the maintenance of RNH1 expression and function. The regulation of E2F1 on the functions of RNH1 in LUAD cells was explored. Mouse experiments were performed to confirm the in-vivo role of RNH1 in LUAD. mRNA sequencing indicated that RNH1 overexpression altered the expression profile of LUAD cells. RESULTS: RNH1 expression in LUAD tissues of patients was presented in this work. Importantly, RNH1 knockdown improved the proliferation, migration and invasion abilities of cells and RNH1 overexpression produced the opposite effects. Dual luciferase assay proved that E2F1 bound to the RNH1 promoter (-1064 ∼ -1054, -1514 ∼ -1504) to reduce the transcriptional activity of RNH1. ChIP assay indicated that E2F1 DNA was enriched at the RNH1 promoter (-1148 ∼ -943, -1628 ∼ -1423). Pull-down assays also showed the association between E2F1 and RNH1 promoter (-1148 ∼ -943). E2F1 overexpression contributed to the malignant behavior of LUAD cells, while RNH1 overexpression reversed it. High-throughput sequencing showed that RNH1 overexpression induced multiple genes expression changes, thereby modulating LUAD-related processes. CONCLUSION: Our study demonstrates that binding of E2F1 to the RNH1 promoter may lead to inhibition of RNH1 expression and thus promoting the development of LUAD.

Absence of E2f1 Negates Pro-osteogenic Impacts of p21 Absence.

Loss of p21 leads to increased bone formation post-injury; however, the mechanism(s) by which this occurs remains undetermined. E2f1 is downstream of p21 and as a transcription factor can act directly on gene expression; yet it is unknown if E2f1 plays a role in the osteogenic effects observed when p21 is differentially regulated. In this study we aimed to investigate the interplay between p21 and E2f1 and determine if the pro-regenerative osteogenic effects observed with the loss of p21 are E2f1 dependent. To accomplish this, we employed knockout p21 and E2f1 mice and additionally generated a p21/E2f1 double knockout. These mice underwent burr-hole injuries to their proximal tibiae and healing was assessed over 7 days via microCT imaging. We found that p21 and E2f1 play distinct roles in bone regeneration where the loss of p21 increased trabecular bone formation and loss of E2f1 increased cortical bone formation, yet loss of E2f1 led to poorer bone repair overall. Furthermore, when E2f1 was absent, either individually or simultaneously with p21, there was a dramatic decrease of the number of osteoblasts, osteoclasts, and chondrocytes at the site of injury compared to p21-/- and C57BL/6 mice. Together, these results suggest that E2f1 regulates the cell populations required for bone repair and has a distinct role in bone formation/repair compared to p21-/-E2f1-/-. These results highlight the possibility of cell cycle and/or p21/E2f1 being potential druggable targets that could be leveraged in clinical therapies to improve bone healing in pathologies such as osteoporosis.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.