High E2F1 Research Articles

Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity.

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

Blocking the E2F transcription factor 1/high-mobility group box 2 pathway enhances the intervention effects of α-santalol on the malignant behaviors of liver cancer cells

In view of the tumor-inhibiting effect of α-santalol in various cancers and the role of E2F transcription factor 1 (E2F1) as an important target for anticancer research, this study investigates the relation between α-santalol and E2F1, as well as the effect of α-santalol on liver cancer progression and the corresponding mechanism. Concretely, liver cancer cells were treated with different concentrations of α-santalol. The IC50 value of α-santalol was determined using Probit regression analysis. Then, transcription factors that are targeted by α-santalol and differentially expressed in liver cancer were screened out. The clinicopathological impact of E2F1 and its targets were evaluated and predicted. The expressions of E2F1 and high-mobility group box 2 (HMGB2) and their correlation in the liver cancer tissues were analyzed by bioinformatics. The effects of E2F1 and HMGB2 on the biological characteristics of liver cancer cells were examined through loss/gain-of-function and molecular assays. With the extension of treatment time, the inhibitory effects of 10 μmol/L and 20 μmol/L α-santalol on cancer cell survival rate were enhanced (P < 0.001). E2F1 and HMGB2 were highly expressed and positively correlated in liver cancer tissues (P < 0.05). High E2F1 expression was correlated with large tumors and high TNM stages (P < 0.05). E2F1 knockdown promoted the effects of α-santalol on dose-dependently inhibiting viability, colony formation, invasion and migration (P < 0.05). Moreover, E2F1 knockdown reduced the IC50 value and HMGB2 level, while HMGB2 overexpression produced opposite effects. HMGB2 overexpression and E2F1 knockdown mutually counteracted their effects on the IC50 value and on the viability and apoptosis of α-santalol-treated liver cancer cells (P < 0.01). Collectively, blocking the E2F1/HMGB2 pathway enhances the intervention effects of α-santalol on the proliferation, migration and invasion of liver cancer cells.

TP53/mTORC1-mediated bidirectional regulation of PD-L1 modulates immune evasion in hepatocellular carcinoma

BackgroundImmunotherapy has facilitated great breakthroughs in the treatment of hepatocellular carcinoma (HCC). However, the efficacy and response rate of immunotherapy are limited and vary among different patients with HCC. TP53...

Association of a high E2F targets score with survival in hepatocellular carcinoma patients.

4106 Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Multiple staging systems have all been proposed to date; however, the optimal tool for stratification of prognosis is still not universally accepted. E2F targets are the essential component of cell proliferation, thus we hypothesized that a score to quantify E2F activity is expected to reflect the aggressiveness and prognosis of HCC. Methods: Total of 655 HCC patients from TCGA (The Cancer Genome Atlas) -HCC ( n=358), GSE6764 ( n=75), GSE76427 ( n = 115), and GSE89377 ( n=107) cohorts were analyzed. The score was generated using MSigDb Hallmark E2F Targets gene set using Gene Set Variation Analysis. Results: The E2F targets scores of TGCA-HCC patients showed a bimodal distribution, thus high vs. low score groups were divided by median. As expected, high E2F targets enriched all of the Hallmark cell proliferation-related gene sets; E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic spindle. It also enriched gene sets known to aggravate cancer, such as Glycolysis, mTORC1 signaling, DNA repair, and Unfolded protein response. E2F targets were significantly associated with histological grade, tumor size, and stage, as well as proliferation score and MKI67 expression. Furthermore, a higher E2F targets score was significantly associated with higher intra-tumoral genomic heterogeneity and homologous recombination deficiency, suggesting greater tumor aggressiveness. In agreement, E2F targets score correlated with the pathological progression from normal liver, cirrhosis, dysplasia, to early and advanced HCC consistently in two cohorts (GSE6764 and GSE89377). Further, the abundance of hepatocytes, fibroblasts, adipocytes, and lymphatic endothelial cells were all significantly less in high E2F HCC, which reflects proliferative cancer. On the other hand, there was no relationship between E2F and mutation rates or neoantigens. High E2F was associated with high infiltration of anti-cancerous immune cells; CD8, CD4 memory, and Th1 cells, however, there was no difference in cytolytic activity, and it did not enrich any of immune response-related gene sets. High E2F HCC was associated with worse disease-free (DFS), disease-specific (DSS), and overall survival (OS), and this was the case in both early (I and II) and late (III and IV) stages. A multivariate analysis revealed that the E2F targets score was an independent prognostic factor for OS (HR=1.68, 95%CI= 1.15–2.46, p = 0.007) as well as DSS (HR=1.81, 95%CI=1.27–2.59, p = 0.001) in patients with HCC. Conclusions: We found that the E2F targets score not only indicates cell proliferation, but also is associated with cancer aggressiveness and worse survival. Our findings suggest a possible future use of the E2F targets score as a prognostic biomarker in patients with HCC.

Prognostic stratification of endometrial cancers with high microsatellite instability or no specific molecular profile.

To identify high-risk disease in clinicopathologic low-risk endometrial cancer (EC) with high microsatellite instability (MSI-H) or no specific molecular profile (NSMP) and therapeutic insensitivity in clinicopathologic high-risk MSI-H/NSMP EC. We searched The Cancer Genome Atlas for DNA sequencing, RNA expression, and surveillance data regarding MSI-H/NSMP EC. We used a molecular classification system of E2F1 and CCNA2 expression and sequence variations in POLE, PPP2R1A, or FBXW7 (ECPPF) to prognostically stratify MSI-H/NSMP ECs. Clinical outcomes were annotated after integrating ECPPF and sequence variations in homologous recombination (HR) genes. Data were available for 239 patients with EC, which included 58 MSI-H and 89 NSMP cases. ECPPF effectively stratified MSI-H/NSMP EC into distinct molecular groups with prognostic implications: molecular low risk (MLR), with low CCNA2 and E2F1 expression, and molecular high risk (MHR), with high CCNA2 and E2F1 expression and/or PPP2R1A and/or FBXW7 variants. The 3-year disease-free survival (DFS) rate was 43.8% in the MHR group with clinicopathologic low-risk indicators and 93.9% in the MLR group (P<.001). In the MHR group, wild-type HR genes were present in 28% of cases but in 81% of documented recurrences. The 3-year DFS rate in patients with MSI-H/NSMP EC with clinicopathologic high-risk indicators was significantly higher in the MLR (94.1%) and MHR/HR variant gene (88.9%) groups than in the MHR/HR wild-type gene group (50.3%, P<.001). ECPPF may resolve prognostic challenges for MSI-H/NSMP EC by identifying occult high-risk disease in EC with clinicopathologic low-risk indicators and therapeutic insensitivity in EC with clinicopathologic high-risk indicators.

E2F target score is associated with cell proliferation and survival of patients with hepatocellular carcinoma

BackgroundE2F target genes are essential for the cell cycle. A score that quantifies its activity is expected to reflect the aggressiveness and prognosis of hepatocellular carcinoma. MethodsCohorts of hepatocellular carcinoma patients (total n = 655) from The Cancer Genome Atlas, GSE89377, GSE76427, and GSE6764 were analyzed. The cohorts were divided into high versus low by the median. ResultsAll the Hallmark cell proliferation-related gene sets were consistently enriched in hepatocellular carcinoma with high E2F targets score, and E2F score was associated with grade, tumor size, American Joint Committee on Cancer staging, proliferation score, and MKI67 expression, as well as with less abundance of hepatocytes and stromal cells. E2F targets enriched DNA repair, mTORC1 signaling, glycolysis, and unfolded protein response gene sets and were significantly associated with the higher intratumoral genomic heterogeneity, homologous recombination deficiency, and progression of hepatocellular carcinoma. On the other hand, there was no relationship between E2F targets and mutation rates or neoantigens. High E2F hepatocellular carcinoma did not enrich any of the immune-response–related gene sets but was associated with high infiltration of Th1, Th2 cells, and M2 macrophage; however, there was no difference in cytolytic activity. In both early (I and II) and late (III and IV) stages of hepatocellular carcinoma, a high E2F score was associated with worse survival and was an independent prognostic factor for overall and disease-specific survival in patients with hepatocellular carcinoma. ConclusionThe E2F target score, associated with cancer aggressiveness and worse survival, could be used as a prognostic biomarker in patients with hepatocellular carcinoma.

E2F1 inhibits cellular senescence and promotes oxaliplatin resistance in colorectal cancer.

Doctors have always been overwhelmed by tumor drug resistance because it is a major challenge in the clinical treatment of tumors. Cellular senescence has a strong relationship with the development of tumor drug resistance. Herein, we aimed to explore new regulatory factors involved in the aging process of colorectal cancer (CRC) cells and assess the effect of cellular senescence on CRC drug resistance. Genes associated with cellular senescence for anticipating regulatory factors were first used, and the regulatory molecules of survival significance were then identified based on the results of public database analysis. The effects of E2F translation factor 1 (E2F1) on CRC cell viability, invasion, migration, and cellular senescence processes were assessed through 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), Transwell, scar repairining, β-galactosidase staining, and cell immunofluorescence assays, respectively. Overexpression or silencing plasmids were used for transfecting HCT116 or OXA-HCT116 to assess the effect of E2F1 on the senescence process and drug resistance in CRC cells. On combining the database analysis results with those of our studies, we found that E2F1 was a critical regulator of cellular senescence in CRC. In the in vitro experiments, the E2F1 overexpression significantly stimulated the proliferation, invasion, and migration of CRC cells and even reduced oxaliplatin-induced senescence, further enhancing their resistance to oxaliplatin. Conversely, the tumorigenesis of colorectal cancer was repressed after the suppression of E2F1. Furthermore, CRC cells, which were otherwise resistant to oxaliplatin, also showed senescent phenotypes. Our results suggest that E2F1 suppresses the aging of CRC cells and tumor cells develop resistance to oxaliplatin through high E2F1 expression. Moreover, E2F1 may act as a possible target for oxaliplatin resistance studies.

MiRNA-329-3p suppresses proliferation and metastasis of endometrial carcinoma through downregulating E2F1.

Existing evidences have revealed the crucial roles of E2 promoter binding factor-1 (E2F1) during the tumorigenesis and progression process of multiple human tumors. However, the expression patterns, biological functions, as well as the underlying molecular mechanism of E2F1 in endometrial carcinoma yet remain largely unclear. The expression patterns and clinical prognostic value of E2F1 in endometrial carcinoma were evaluated using bioinformatics methods. Protein and mRNA, miRNA expression levels in tissues and cells were measured using immunohistochemistry, western blotting, and qRT-PCR assays. Cell viability and cell cycle distribution were examined using CCK-8 assay and flow cytometry, respectively. Scratch healing assay and Transwell assay were applied to measure cell migration and invasion ability. Bioinformatic analysis and luciferase reporter assays were conducted to confirm the targeting relationship between E2F1 and miR-329-3p. Moreover, a series of in vitro and in vivo functional experiments were employed to evaluate the effect of the miR-329-3p/E2F1 axis on cell growth and metastasis. Clinically, E2F1 was aberrantly expressed in endometrial carcinoma tissues and was correlated with advanced FIGO stage, histological type, p53 mutation, poor survival, and degree of tumor cell differentiation. ROC curves analysis also reveals that E2F1 has a high AUC value (up to 0.952, 95% CI: 0.915-0.988), indicating the promising diagnostic value of E2F1 level in endometrial carcinoma. In addition, in vitro gain and loss-of-functional experiments verified that high E2F1 can promote cell proliferation, cell cycle, migration, invasion, and EMT process. In-depth mechanism studies revealed that E2F1 was a downstream target gene of miR-329-3p, and miR-329-3p overexpression could effectively abrogate its promotion of cell malignant biological behavior. Collectively, our findings suggested that the miR-329-3p/E2F1 axis plays a crucial role in the progression of endometrial carcinoma, indicating that E2F1 can be considered a promising diagnostic and prognostic biomarker for endometrial carcinoma patients.

ECPPF (E2F1, CCNA2, POLE, PPP2R1A, FBXW7) stratification: Profiling high-risk subtypes of histomorphologically low-risk and treatment-insensitive endometrioid endometrial cancer.

In endometrial cancer, occult high-risk subtypes (rooted in histomorphologically low-risk disease) with insensitivity to adjuvant therapies impede improvements in therapeutic efficacy. Therefore, we aimed to assess the ability of molecular high-risk (MHR) and low-risk (MLR) ECPPF (E2F1, CCNA2, POLE, PPP2R1A, FBXW7) stratification to profile recurrence in early, low-risk endometrioid endometrial cancer (EEC) and insensitivity to platinum-based chemotherapy or radiotherapy (or both) in high-risk EEC. Using The Cancer Genome Atlas endometrial cancer database, we identified 192 EEC cases with available DNA sequencing and RNA expression data. Molecular parameters were integrated with clinicopathologic risk factors and adverse surveillance events. MHR was defined as high (-H) CCNA2 or E2F1 log2 expression (≥2.75), PPP2R1A mutations (-mu), or FBXW7mu; MLR was defined as low (-L) CCNA2 and E2F1 log2 expression (<2.75). We assessed 164 cases, plus another 28 with POLEmu for favorable-outcomes comparisons. MHR and MLR had significantly different progression-free survival (PFS) rates (P < .001), independent of traditional risk factors (eg, TP53mu), except for stage IV disease. PFS of CCNA2-L/E2F1-L paralleled that of POLEmu. ECPPF status stratified responses to adjuvant therapy in stage III-IV EEC (P < .01) and profiled stage I, grade 1-2 cases with risk of recurrence (P < .001). MHR was associated with CTNNB1mu-linked treatment failures (P < .001). Expression of homologous recombination repair (HR) and cell cycle genes was significantly elevated in CCNA2-H/E2F1-H compared with CCNA2-L/E2F1-L (P<1.0E-10), suggesting that HR deficiencies may underlie the favorable PFS in MLR. HRmu were detected in 20.7%. No treatment failures were observed in high-grade or advanced EEC with HRmu (P = .02). Favorable PFS in clinically high-risk EEC was associated with HRmu and MLR ECPPF (P < .001). In summary, MLR ECPPF and HRmu were associated with therapeutic efficacy in EEC. MHR ECPPF was associated with low-risk, early-stage recurrences and insensitivity to adjuvant therapies.

E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized “anti-H2AZ2” query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing “enhanceosomes” that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.

Clinical Implication of E2F Transcription Factor 1 in Hepatocellular Carcinoma Tissues.

Background: To date, the clinical management of advanced hepatocellular carcinoma (HCC) patients remains challenging and the mechanisms of E2F transcription factor 1 (E2F1) underlying HCC are obscure. Materials and Methods: Our study integrated datasets mined from several public databases to comprehensively understand the deregulated expression status of E2F1. Tissue microarrays and immunohistochemistry staining was used to validate E2F1 expression level. The prognostic value of E2F1 was assessed. In-depth subgroup analyses were implemented to compare the differentially expressed levels of E2F1 in HCC patients with various tumor stages. Functional enrichments were used to address the predominant targets of E2F1 and shedding light on their potential roles in HCC. Results: We confirmed the elevated expression of E2F1 in HCC. Subgroup analyses indicated that elevated E2F1 level was independent of various stages in HCC. E2F1 possessed moderate discriminatory capability in differentiating HCC patients from non-HCC controls. Elevated E2F1 correlated with Asian race, tumor classification, neoplasm histologic grade, eastern cancer oncology group, and plasma AFP levels. Furthermore, high E2F1 correlated with poor survival condition and pooled HR signified E2F1 as a risk factor for HCC. Enrichment analysis of differentially expressed genes, coexpressed genes, and putative targets of E2F1 emphasized the importance of cell cycle pathway, where CCNE1 and CCNA2 served as hub genes. Conclusions: We confirmed the upregulation of E2F1 and explored the prognostic value of E2F1 in HCC patients. Two putative targeted genes (CCNE1 and CCNA2) of E2F1 were identified for their potential roles in regulating cell cycle and promote antiapoptotic activity in HCC patients.

A model for the regulation of apoptosis intrinsic pathway: The potential role of the transcriptional regulator E2F in the point of no return

Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This “Extended Legewie Model” (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.

Cancer Essential Genes Stratified Lung Adenocarcinoma Patients with Distinct Survival Outcomes and Identified a Subgroup from the Terminal Respiratory Unit Type with Different Proliferative Signatures in Multiple Cohorts.

Simple SummarySeveral genes are essential for tumor growth and predict poor prognoses of patients, however, most of their roles in lung adenocarcinoma (LUAD) are unclear. In addition, a good classification strategy for cancer patients would be a useful tool for future personalized medicine. In LUAD, the existing subtype classification, including the terminal respiratory unit (TRU), proximal-inflammatory (PI), and proximal-proliferative (PP) subtypes, is mainly based on genes with variant expression levels across patients without considering the oncogenetic roles of those genes. Thus, the LUAD essential genes were identified and used to stratify patients into distinct survival outcomes, TP53 mutation statuses, E2F target activities, and tumor mutation burdens. Moreover, TRU-type patients could be further divided into clinically and molecularly different subgroups based on our classifier. Integration of existing subtypes with our classification strategy provides a more comprehensive understanding of the heterogeneity of LUAD, and can guide us to identifying potential targets for future personalized medicine.Background: Heterogeneous features of lung adenocarcinoma (LUAD) are used to stratify patients into terminal respiratory unit (TRU), proximal-proliferative (PP), and proximal-inflammatory (PI) subtypes. A more-accurate subtype classification would be helpful for future personalized medicine. However, these stratifications are based on genes with variant expression levels without considering their tumor-promoting roles. We attempted to identify cancer essential genes for LUAD stratification and their clinical and biological differences. Methods: Essential genes in LUAD were identified using genome-scale CRIPSR screening of RNA sequencing data from Project Achilles and The Cancer Genome Atlas (TCGA). Patients were stratified using consensus clustering. Survival outcomes, genomic alterations, signaling activities, and immune profiles within clusters were investigated using other independent cohorts. Findings: Thirty-six genes were identified as essential to LUAD, and there were used for stratification. Essential gene-classified clusters exhibited distinct survival rates and proliferation signatures across six cohorts. The cluster with the worst prognosis exhibited TP53 mutations, high E2F target activities, and high tumor mutation burdens, and harbored tumors vulnerable to topoisomerase I and poly(ADP ribose) polymerase inhibitors. TRU-type patients could be divided into clinically and molecularly different subgroups based on these essential genes. Conclusions: Our study showed that essential genes to LUAD not only defined patients with different survival rates, but also refined preexisting subtypes.

Abstract PS4-35: The E2F cell cycle pathway score to predict treatment response among patients with breast cancer

Abstract Cell cycle progression is a critical component of cell proliferation, and continuous proliferation is one of the hallmarks of cancer. Components regulating the E2F pathway have been identified in nearly every human malignancy and many of them including E2F transcription factors themselves play major roles in cancer progression, metastasis and treatment response of breasts cancer. Their activity therefore is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3,905 tumors of nine breast cancer cohorts for this activity based on their 200 gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had increased expression of cell proliferation-related genes, including G2M checkpoint, MYC targets v1 and v2, MITOTIC spindle, MTORC1 signaling, UNFOLDED protein response, and DNA repair in both the TCGA and METABRIC cohorts (false discovery rate &amp;lt; 0.001 in both cohorts). A high score was significantly associated with greater MKI67 expression (p &amp;lt; 0.001 in both cohorts), histological grade (p &amp;lt; 0.001 in both cohorts), and AJCC pathological stage (p &amp;lt; 0.001 in both cohorts). And Indel and single nucleotide variation neoantigen loads were associated with a high E2F pathway score (p = 0.006 and 0.047 respectively). Furthermore, the E2F pathway score correlated positively with copy number alteration (Spearman r = 0.55, p &amp;lt; 0.001). Intra-tumoral genome heterogeneity and proliferation score were significantly associated with the E2F pathway score as well (p &amp;lt; 0.001). The high E2F pathway score group demonstrated significantly higher fractions of not only pro-cancerous regulatory T cells, helper T cell (Th2), but also anti-cancerous CD4 memory T cell, helper T cell (Th1), and M1 macrophage as well as B cells compared to the low score group in the TCGA cohort (p &amp;lt; 0.001). Similar trends were observed in the METABRIC cohort. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose especially luminal and normal subtype (p = 0.006 and p &amp;lt; 0.001). Interestingly, metastases with a high E2F score were associated with significantly worse PFS in the whole cohort, as well as patient sub-groups with local recurrence and with liver metastasis (p = 0.018, p = 0.025 and p = 0.027). Additionally, the E2F pathway score was significantly decreased with good response to chemotherapy (p &amp;lt; 0.001) and demonstrated higher pCR rate after neoadjuvant therapies in the high E2F pathway score group of estrogen receptor (ER)-positive/HER2-negative cancer (p &amp;lt; 0.001). The E2F score was significantly associated with expression of cyclin-dependent kinase (CDK)-related genes, including CCNE1, CDKN2A, CDKN2D, CDK2, CDK4 and CDK6 (all p &amp;lt; 0.001), and immune checkpoint molecule-related genes including PD-L1/2, CTLA4, IDO1, LAG3 and TIGIT (p = 0.013, 0.014, &amp;lt; 0.001, &amp;lt; 0.001, &amp;lt; 0.001 and 0.002, respectively). These results were validated by other cohort. Finally, the E2F score was strongly correlated with sensitivity to CDK inhibition in ER+/HER2- breast cancer cell-lines (Spearman r = 0.90, p = 0.04). In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts responsiveness of ER+/HER2- patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors. Citation Format: Masanori Oshi, Hideo Takahashi, Yoshihisa Tokumaru, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. The E2F cell cycle pathway score to predict treatment response among patients with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-35.

Identification of Differential Tumor Subtypes of T1 Bladder Cancer

Stage T1 bladder cancers have the highest progression and recurrence rates of all non–muscle-invasive bladder cancers (NMIBCs). Most T1 cancers are treated with bacillus Calmette-Guérin (BCG), but many will progress or recur, and some T1 patients will die from bladder cancer. Particularly aggressive tumors could be treated with early cystectomy. To better understand the molecular heterogeneity of T1 cancers, we performed transcriptome profiling and unsupervised clustering, and identified five consensus subtypes of T1 tumors treated with repeat transurethral resection (reTUR) and induction and maintenance BCG. The T1-LumGU subtype was associated with carcinoma in situ (CIS; six/13, 46% of all CIS), had high E2F1 and EZH2 expression, and was enriched in E2F target and G2M checkpoint hallmarks. The T1-Inflam subtype was inflamed and infiltrated with immune cells. While most T1 tumors were classified as luminal papillary, the T1-TLum subtype had the highest median luminal papillary score and FGFR3 expression, no recurrence events, and the fewest copy number gains. T1-Myc and T1-Early subtypes had the most recurrences (14/30 within 24 mo), the highest median MYC expression, and, when combined, had significantly worse recurrence-free survival than the other three subtypes. T1-Early had five (38%) recurrences within the first 6 mo of BCG, and repressed IFN-α and IFN-γ hallmarks and inflammation. We developed a single-patient T1 classifier and validated our subtype biology in a second cohort of T1 tumors. Future research will be necessary to validate the proposed T1 subtypes and to determine if therapies can be individualized for each subtype. Patient summaryWe identified and characterized expression subtypes of high-grade stage T1 bladder cancer that are biologically heterogeneous and have variable responses to bacillus Calmette-Guérin treatment. We validated the subtypes and describe a single-patient classifier.

The E2F Pathway Score as a Predictive Biomarker of Response to Neoadjuvant Therapy in ER+/HER2- Breast Cancer.

E2F transcription factors play critical roles in the cell cycle. Therefore, their activity is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3905 tumors of nine breast cancer cohorts for this activity based on their gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had an increased expression of cell proliferation-related genes. A high score was significantly associated with shorter patient survival, greater MKI67 expression, histological grade, stage, and genomic aberrations. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose. Although tumors with a high score had greater infiltration by both pro- and anti-cancerous immune cells, they had an increased expression of immune checkpoint genes. Estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative cancer with a high E2F score achieved a significantly higher pathological complete response (pCR) rate to neoadjuvant chemotherapy. The E2F score was significantly associated with the expression of cyclin-dependent kinase (CDK)-related genes and strongly correlated with sensitivity to CDK inhibition in cell lines. In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts the responsiveness of ER-positive/HER2-negative patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors.

E2F cell cycle pathway score as a predictive biomarker of ER+/HER2- breast cancer response to neoadjuvant chemotherapy.

e12593 Background: Although the survival rate of patients diagnosed with breast cancer has greatly improved over the last decades, the metastatic stage of the disease remains incurable. Estrogen receptor positive (ER+)/human receptor 2 negative (HER2-) breast cancers accounts for approximately 70% of metastatic breast cancers and, it is responsible for most of the deaths from the disease. Another issue of ER+/HER2- breast cancer is its poor response to neoadjuvant chemotherapy (NAC). Recently, several clinical trials have shown that endocrine therapy plus FDA-approved cyclin-dependent kinases (CDK) 4/6 inhibitors have improved the treatment response to patients with advanced ER+/HER2 breast. The CDK 4/6 is an important E2F-related factor. Therefore, we hypothesized that E2F pathway is predictive biomarker for treatment response of ER+/HER2- breast cancer. Methods: METABRIC dataset was used for training cohort. And TCGA breast cancer as well as four neoadjuvant therapeutic cohorts were used as validation cohort. The E2F pathway score was calculated by Gene Set Variant Analysis (GSVA). Results: Tumors with high E2F pathway score enriched cell proliferation and cell cycle pathway related gene sets (G2M, MYC v1, and v2, MITOTIC, MTORC1, PI3K AKT MTOR, and DNA repair; FDR &lt; 0.001). The high E2F pathway score was significantly associated with TNBC and HER2+ (p &lt; 0.001), which are known to be clinically aggressive, and advanced stage (p &lt; 0.001) and high grade (p &lt; 0.001). The score also correlated with copy mutation (r = 0.605, p &lt; 0.01). The patients, who had early recurrence before 5 years, had significantly high score rather than non-recurrence patients (p &lt; 0.001). Moreover, these results were validated in TCGA cohort. The high E2F pathway score group had a higher pCR rate than the low score group with ER+/HER2- breast cancer (p &lt; 0.001). Interestingly, the tumors in high score group expressed higher CDK pathway related molecules compared to low score group. Conclusions: The E2F pathway score was found to be a predictive biomarker for neoadjuvant chemotherapy in ER+/HER2- breast cancer patients.

Protein expression profiles and prognostic value of E2F family members in clear cell renal cell carcinoma

The derangement of the cell cycle facilitates uncontrolled cell proliferation and acquisition of genetic alterations favorable for malignancy. However, the protein expression profiles of E2 F family cell cycle regulators in clear cell renal cell carcinoma (ccRCC) have not yet been thoroughly investigated. In this study, we aimed to examine the protein expression profiles and prognostic value of E2 F1, E2 F3, and E2 F4 in ccRCC cases. The immunohistochemical expression of E2 F1, E2 F3, and E2 F4 was quantitatively scored in 180 ccRCC tumor tissues and 79 normal kidney tissues. The prognostic implications of these E2 F members were determined. We found that ccRCC tumor cells showed higher nuclear expression of E2 F1, E2 F3 and E2 F4 than normal kidney samples. High E2 F1 and E2 F3 expression in tumor cells was associated with poor prognostic factors of ccRCC, whereas high E2 F4 correlated with beneficial prognostic factors. High expression of E2 F1 and E2 F3 in tumor cells was correlated with a poor overall and recurrence-free survival, while high E2 F4 expression did not. In conclusion, E2 F1, E2 F3 and E2 F4 may function as oncogenes during tumorigenesis of ccRCC, although they contribute to the progression of ccRCC in different ways. Additional studies are required to clarify the conflicting role of E2 F4 in the tumor evolution of ccRCC.

Reciprocal regulation of miR-1205 and E2F1 modulates progression of laryngeal squamous cell carcinoma

The burgeoning functions of many microRNAs (miRs) have been well study in cancer. However, the level and function of miR-1205 in laryngeal squamous cell cancer remains unknown. In the current research, we validated that miR-1205 was notably downregulated in human laryngeal squamous cell carcinoma (LSCC) samples in comparison with tissues adjacent to LSCC, and correlated with T stage, lymph node metastasis, and clinical stage. Using Kaplan–Meier analysis indicates that high expression of miR-1205 has a favorable prognosis for patients with LSCC. Functional assays show that enforced miR-1205 expression attenuates the migration, growth, and invasion of LSCC cells. And E2F1 is verified to be a target of miR-1205, while E2F1 binds to miR-1205 promoter and transcriptionally inhibits miR-1205 expression. Overexpression of E2F1 reverses the inhibitory impacts of miR-1205 on LSCC cells in part. Importantly, E2F1 is abnormally increased in LSCC tissues, and its protein levels were inversely relevant to miR-1205 expression. High E2F1 protein level is in connection with clinical stage, T stage, lymph node metastasis, and poor prognosis. Consequently, reciprocal regulation of miR-1205 and E2F1 plays a crucial role in the progression of LSCC, suggesting a new miR-1205/E2F1-based clinical application for patients of LSCC.

Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness

BackgroundBladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.MethodsRT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.ResultsHerein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a ‘high E2F1/APLF/DCLRE1C’ signature, which was found to be particularly favorable for BC patient survival.ConclusionSuppression of the ‘out-of-context’ activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.