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Ferroptosis, a unique form of nonapoptotic-regulated cell death caused by overwhelming lipid peroxidation, represents an emerging tumor suppression mechanism. Growing evidence has demonstrated that cell metabolism plays an important role in the regulation of ferroptosis. Specifically, the association between methionine metabolism and ferroptosis remains undefined.We performed in vitro and in vivo experiments to evaluate the influence of methionine metabolism on ferroptosis sensitivity. Pharmacological and genetic blockade of the methionine cycle was utilized and relevant molecular analyses were performed.We identified MAT2A as a driver of ferroptosis resistance. Mechanistically, MAT2A mediates the production of S-adenosylmethionine (SAM), which upregulates ACSL3 by increasing the trimethylation of lysine-4 on histone H3 (H3K4me3) at the promoter area, resulting in ferroptosis resistance.Collectively, these results established a link between methionine cycle activity and ferroptosis vulnerability in gastric cancer.

BackgroundPeritoneal dissemination (PD) is the most common mode of metastasis for advanced gastric cancer (GC) with poor prognosis. It is of great significance to accurately predict preoperative PD and develop optimal treatment strategies for GC patients. Our study assessed the diagnostic potential of serum tumor markers and clinicopathologic features, to improve the accuracy of predicting the presence of PD in GC patients.MethodsIn our study, 1264 patients with GC at Fudan University Shanghai Cancer Center and Wenzhou people’s hospital from 2018 to 2020 were retrospectively analyzed, including 316 cases of PD and 948 cases without PD. All patients underwent enhanced CT scan or magnetic resonance imaging (MRI) before surgery and treatment. Clinicopathological features, including tumor diameter and tumor stage (depth of tumor invasion, nearby lymph node metastasis and distant metastasis), were obtained by imaging examination. The independent risk factors for PD were screened through univariate and multivariate logistic regression analyses, and the results were expressed with 95% confidence intervals (CIs). A model of PD diagnosis and prediction was established by using Cox proportional hazards regression model of training set. Furthermore, the accuracy of the prediction model was verified by ROC curve and calibration plots.ResultsUnivariate analysis showed that PD in GC was significantly related to tumor diameter (odds ratio (OR)=12.06, pConclusionThe nomogram model constructed using CA125, CA72-4 and depth of invasion increases the accuracy and sensitivity in predicting the incidence of PD in GC patients and can be used as an important tool for preoperative diagnosis.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Background The systemic inflammation score (SIS), as calculated from preoperative serum albumin level and lymphocyte-to-monocyte ratio, has been demonstrated to be a prognostic marker in cancer. The present study intended to investigate the prognostic role of SIS in gastric cancer patients after curative gastrectomy in comparison with other prognostic markers. Methods Preoperative SIS was retrospectively calculated in patients who underwent curative gastrectomy between 2007 and 2011 in Fudan University Shanghai Cancer Center. The prognostic accuracy of each score was compared utilizing time-dependent receiver operating characteristics analysis. Results The higher SIS score was associated with older age, larger tumour size, a more advanced tumour-nodes-metastasis stage and lymph node status, deeper tumour invasion, the presence of lymphovascular invasion and a poorer overall survival and disease-free survival. In time-dependent receiver operating characteristics analysis, the SIS had a higher area under the curve for the prediction of 5-year overall survival than the neutrophil lymphocyte ratio. The SIS maintained the predictive accuracy superiority throughout the observation period. Conclusion The SIS is a useful prognostic marker in gastric cancer patients after curative gastrectomy.

Introduction With the rapid development of minimally invasive surgery, laparoscopic (LAP) wedge resection has become the first-choice treatment for primary gastrointestinal stromal tumors (GISTs) of the stomach. Aim To investigate the safety and feasibility of LAP wedge resection and the choice of surgical treatment for GISTs of the stomach through a meta-analysis and systematic review. Material and methods The literature was widely searched for comparative studies on open (OPEN) and LAP wedge resection for GISTs published before April 2017. The articles were selected after quality assessment. Results Ten reports met the inclusion criteria, with a total sample size of 485 cases. The operation time was similar between the 2 groups (weighted mean difference (WMD): 8.67 min, 95% confidence interval (CI): –8.60 to 25.94, p = 0.33). However, LAP resulted in less blood loss (WMD –32.20 ml, 95% CI: –56.15 to –8.26, p < 0.01), earlier time to flatus (WMD –1.48 days, 95% CI: –1.90 to –1.06, p < 0.01) and to an oral diet (WMD –1.50 days, 95% CI: –2.25 to –0.47, p < 0.01), shorter hospital stay (WMD –2.03 days, 95% CI: –2.68 to –1.38, p < 0.01), and a decreased overall complication rate (relative risk: 0.48, 95% CI: 0.25–0.89, p = 0.01) compared with OPEN. Moreover, long-term follow-up findings indicated no obvious difference between the 2 groups. Conclusions The use of LAP wedge resection for suitable cases is safe and feasible because it causes less blood loss and fewer overall complications and enables faster recovery.

BackgroundThe complement system acts as an integral part of the innate immune response, which acts primarily to remove pathogens and injured cells. Emerging evidence has shown the activation of the immune regulatory function of complements in the tumor microenvironment (TME). We revealed the expression levels of various complements in human cancers and their role in tumor prognosis and immune infiltration.MethodsThe differential expression of complements was exploredviathe Tumor Immune Estimation Resource (TIMER) site and the Oncomine database. To investigate whether these differentially expressed complements have correlation with the prognosis of gastric cancer (GC) and colon cancer, their impact on survival was assessed using the PrognoScan database and Kaplan-Meier plotter. The correlations between complements and tumor immune-infiltrating levels and immune gene markers were statistically explored in TIMER based on Spearman’s correlation coefficients andp-values.ResultsIn two colon cancer cohorts, an increased expression level of DAF (CD55) has statistically significant correlation with poor disease-free survival (DFS). High C3, CR4, and C5aR1 expression levels were significantly related with poor prognosis in GC patients. In addition, C3, CR4, and C5aR1 expression was positively related to the tumor purity and infiltration levels of multiple immune cells in stomach adenocarcinoma (STAD). Moreover, the expression levels of C3, CR4, and C5aR1 were also strongly correlated with various immune marker sets, such as those of tumor-associated macrophages (TAMs), M1 and M2 macrophages, T cell exhaustion, Tregs, and DCs, in STAD. Additionally, CD55 has positive correlation with few immune cell infiltration levels in colon adenocarcinoma (COAD), but its correlation with immune marker sets was not statistically significant.ConclusionThese findings confirm the relationship between various complements and tumor prognosis and immune infiltration in colon cancer and GC. CD55 may serve as an indicator on the survival prognosis of patients with colon cancer. Furthermore, as biomarkers for poor prognosis in GC, complements C3, CR4, and C5aR1 may provide potential biological targets for GC immunotherapy.

Background The aim of this study was to combine clinicopathologic variables associated with overall survival and disease-free survival after curative resection for hepatocellular carcinoma (HCC) with portal vein tumour thrombus (PVTT) into a prediction nomogram. Methods We retrospectively analysed 358 patients who underwent curative resection for HCC with PVTT at the Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China. Two-thirds of the patients were randomly assigned to the training set (n = 252) and one-third were assigned to the validation set (n = 106). Multivariate analysis by Cox proportional hazards regression was performed using the training set, and the nomogram was constructed. Discrimination and calibration were performed using the validation set. Results The multivariate Cox model identified alpha fetoprotein, hepatitis B surface antigen (HBsAg), tumour diameter, tumour capsule, PVTT type and TNM stage as covariates associated with 1-year survival, and alpha fetoprotein, HBsAg, tumour diameter, tumour capsule and PVTT type with half-year disease-free survival. In the validation set, the nomogram exhibited superior discrimination power (Harrell's C-index 0.78) compared with the American Joint Committe on Cancer TNM classification, the Cancer of the Liver Italian Program grade and the Japan Integrated Staging grade. Calibration of the nomogram-predicted survival corresponding closely with the actual survival, the predicted survival was within a 10% margin of ideal nomogram. Conclusion We developed a nomogram predicting 1-year overall survival and half-year disease-free survival after curative resection for HCC with PVTT. Validation data sets revealed good discrimination and calibration, suggesting good clinical utility. The nomogram improved individualized predictions of survival.

Introduction Liver cancer is one of the most common malignancies of the digestive system. Minimally invasive ablation procedures have become one of the major means for treating unresectable multifocal liver cancer and have been extensively applied in primary and metastatic liver cancer treatment. Laparoscopic B-ultrasound-guided microwave ablation is an example of the progress made in this field. Aim To analyze and summarize the results of and experience with laparoscopic B-ultrasound-guided microwave ablation for multifocal primary liver cancer; moreover, the ablation effects were compared between tumors of different sizes. Material and methods Laparoscope-guided needle ablation was conducted on 84 lesions from 32 patients with primary liver cancer based on tumor size, quantity, and location. Moreover, the perioperative data, ablation effects according to tumor size, and long-term follow-up results were analyzed. Results Among the 84 nodules treated via microwave ablation, tumors measuring ≤ 3 cm demonstrated complete ablation upon imaging analysis conducted 1 month after surgery. Moreover, 5 of the tumors measuring > 3 cm demonstrated incomplete ablation. In these cases, a second procedure was performed, until imaging studies confirmed that complete ablation was achieved. Conclusions Laparoscopic microwave ablation allows for precise puncture positioning, an effective ablation range, and safe and feasible surgery, which is especially suitable for liver tumors located in sites difficult to access.

Background Hepatocellular carcinoma (HCC) represents one of the most common malignancies worldwide. In two pubic long noncoding RNA (lncRNA) profiling studies of HCC, linc00462 was consistently upregulated. We analyzed the clinical significance and biological role of linc00462 in HCC. Methods We performed quantitative real-time PCR analysis to determine the levels of linc00462 in HCC tissues from 49 patients. Functional analysis was performed in cell lines and in an animal model to support clinical findings. Results Our data showed that linc00462 was significantly upregulated in HCC tissues compared with matched normal tissues. The knockdown of linc00462 in HCC cells resulted in a much less aggressive oncogenic phenotype, and linc00462 downregulation contribute to the inactivation of the PI3K/AKT signaling pathway. Conclusions linc00462 may be a potential therapeutic target in HCC.

Hepatocellular carcinoma (HCC) is among the most frequently observed forms of cancer. MicroRNAs (miRNAs) are increasingly thought to play a key role in regulating the onset and progression of a wide range of cancer types. In the present report, we found that miR-455-5p expression was significantly decreased in both HCC patient tumor tissues and cell lines, and that this reduction in expression was linked to poorer patient outcomes. When we overexpressed miR-455-5p in HCC cell lines (Huh7 and HepG2), this was linked with impaired proliferation, colony formation, migration, and invasion. We further found that this miRNA was able to directly bind the insulin growth factor receptor (IGF-1R) 3'-untranslated region, thereby suppressing IGF-1R expression in HCC cells. Consistent with this, miR-455-5p overexpression was associated with reduced glucose transporter (GLUT) 1 expression, which in turn inhibited HCC cell uptake of glucose, production of lactate, and generation of ATP. Together these results thus indicate that mIR-455-5p is able to suppress tumor functionality via impairing glycolysis in HCC cells, highlighting this miRNA as a potential target for anti-cancer therapeutic interventions.

Gastric cancer (GC) remains a threat to public health with high incidence and mortality worldwide. Increasing evidence demonstrates that long non‐coding RNAs (lncRNAs) play critical regulatory roles in cancer biology, including GC. Previous profiling study showed that lncRNA linc00261 was aberrantly expressed in GC. However, the role of linc00261 in GC progression and the precise molecular mechanism remain unknown. In this study, we report that linc00261 was significantly down‐regulated in GC tissues and the expression level of linc00261 negatively correlated with advanced tumour status and clinical stage as well as poor prognostic outcome. In vitro functional assays indicate that ectopic expression of linc00261 suppressed cell invasion by inhibiting the epithelial–mesenchymal transition (EMT). By RNA pull‐down and mass spectrum experiments, we identified Slug as an RNA‐binding protein that binds to linc00261. We confirmed that linc00261 down‐regulated Slug by decreasing the stability of Slug proteins and that the tumour‐suppressive function of linc00261 can be neutralized by Slug. linc00261 may promote the degradation of Slug via enhancing the interaction between GSK3β and Slug. Moreover, linc00216 overexpression repressed lung metastasis in vivo. Together, our findings suggest that linc00261 acts a tumour suppressor in GC by decreasing the stability of Slug proteins and suppressing EMT. By clarifying the mechanisms underlying GC progression, these findings may facilitate the development of novel therapeutic strategies for GC.

Thyroid cancer is one of the most common carcinomas of the endocrine system with an increasing incidence. A growing number of studies have focused on the diagnostic and prognostic values of dysregulated microRNAs (miRNAs) in thyroid carcinoma. However, differences in the measurement platforms, variations in lab protocols, and small sample sizes can make gene profiling data incomparable. A meta‐review of the published studies that compared miRNA expression data of thyroid carcinoma and paired normal tissues was performed to identify potential miRNA biomarkers of thyroid carcinoma with the vote‐counting strategy. Two hundred and thirty‐six aberrantly expressed miRNAs were reported in 19 microRNA expression profiling studies. Among them, 138 miRNAs were reported in at least two studies. We also provided a meta‐signature of differentially expressed miRNAs between individual histological types of thyroid carcinoma and normal tissues. The experimental validation with qRT‐PCR analysis verified that the profiles identified with the meta‐review approach could effectively discriminate papillary thyroid carcinoma tissues from paired noncancer tissues. The meta‐review of miRNA expression profiling studies of thyroid carcinoma would provide information on candidate miRNAs that could potentially be used as biomarkers in thyroid carcinoma.

Long noncoding RNAs (lncRNAs) have been demonstrated to play a role in carcinogenesis, but their mechanisms of function remain elusive. We explored the mechanisms of the oncogenic role of GCAWKR in gastric cancer (GC) using human tissues and cell lines. The in situ hybridization analysis was utilized to determine GCAWKR levels in samples from 42 GC patients and real-time qPCR in tissues from 123 patients. The GCAWKR levels were modulated in GC cell lines, and relevant biological and molecular analyses were performed. Levels of the GCAWKR were upregulated in GC tissues compared with normal tissues and associated with tumor size, lymph node metastasis, TNM stage, and patient outcomes. GCAWKR affected cell proliferation and cell invasion in multiple GC models. Mechanistically, GCAWKR bound WDR5 and KAT2A and acted as a molecular scaffold of WDR5/KAT2A complexes, modulating the affinity for WDR5/KAT2A complexes in the target gene's promoter region. Thus, our data defined a mechanism of lncRNA-mediated carcinogenesis in GC, suggesting new therapeutic targets in GC.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to alarge cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-bindingelements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

Background: It is known that inflammation promotes cancer development. However, a few studies have evaluated the prognostic significance of inflammatory biomarkers in gastric cancer (GC). Methods: In this study, 2,334 patients who underwent gastrectomy for GC at Fudan University Shanghai Cancer Center between 2003 and 2007 were retrospectively analyzed, and 1,227 patients were found to be eligible. The preoperative serum neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio (LMR), and albumin/globulin ratio (AGR) levels were analyzed. A nomogram was constructed with the Cox proportional hazards regression model in the training set (n = 818) to predict the probability of overall survival (OS) and disease-free survival (DFS). The predictive accuracy and discriminative ability were determined using the concordance index (C-index) and calibration curve. Results: We found that lower AGR and LMR values were correlated with decreased OS, lower LMR values, and higher NLR values with a decreased DFS. Other significant factors were included to construct the nomogram. The discriminative ability of the nomogram was higher than that of the eighth American Joint Committee on Cancer tumor-node-metastasis (TNM) staging system (0.746 for TNM v.s. 0.654 for the nomogram, p < 0.001). Conclusions: The nomogram yielded a more accurate prognostic prediction in GC patients after gastrectomy, suggesting great clinical value.

The aim of this study was to combine clinicopathologic variables associated with overall survival and disease-free survival after curative resection for hepatocellular carcinoma (HCC) with portal vein tumour thrombus (PVTT) into a prediction nomogram.We retrospectively analysed 358 patients who underwent curative resection for HCC with PVTT at the Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China. Two-thirds of the patients were randomly assigned to the training set (n = 252) and one-third were assigned to the validation set (n = 106). Multivariate analysis by Cox proportional hazards regression was performed using the training set, and the nomogram was constructed. Discrimination and calibration were performed using the validation set.The multivariate Cox model identified alpha fetoprotein, hepatitis B surface antigen (HBsAg), tumour diameter, tumour capsule, PVTT type and TNM stage as covariates associated with 1-year survival, and alpha fetoprotein, HBsAg, tumour diameter, tumour capsule and PVTT type with half-year disease-free survival. In the validation set, the nomogram exhibited superior discrimination power (Harrell's C-index 0.78) compared with the American Joint Committe on Cancer TNM classification, the Cancer of the Liver Italian Program grade and the Japan Integrated Staging grade. Calibration of the nomogram-predicted survival corresponding closely with the actual survival, the predicted survival was within a 10% margin of ideal nomogram.We developed a nomogram predicting 1-year overall survival and half-year disease-free survival after curative resection for HCC with PVTT. Validation data sets revealed good discrimination and calibration, suggesting good clinical utility. The nomogram improved individualized predictions of survival.

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

Flow cytometric analysis of Annexin V staining. Figure S2 MALAT1 expression levels vary across different cancer types in the TCGA database. Figure S3 (a) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transduction of lentiviruses encoding MALAT1 shRNA or a scrambled shRNA. Northern blot analysis of MALAT1 expression in SGC7901/VCR cells after transduction of lentiviruses encoding MALAT1 shRNA or a scrambled shRNA. (b) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transfection of lentivirus harboring the full-length human MALAT1 sequence or the empty vector. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. *, p < 0.05. Figure S4 MALAT1 promotes autophagy. (a) SGC7901/VCR cells stably transfected with full-length human MALAT1 sequence or the empty vector were subjected to Western blot analysis of LC3-II and p62. (b) Autophagy was evaluated using transmission electron microscopy in SGC7901/VCR stably transfected with full-length human MALAT1 sequence or the empty vector. (c) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant upregulation of MALAT1 as determined with qRT-PCR analysis. (d) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant activation of autophagy, while MALAT1 knockdown blunted the autophagic response to cisplatin. (e) SGC7901 cells transfected with full-length human MALAT1 sequence or the empty vector were treated with the cisplatin (5 μg/ml) for 24 h, caspase-3 protein was detected by Western blot. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. *, p < 0.05. (f) SGC7901/VCR cells stably transfected with shRNA-lncRNA-ATB or a control were subjected to Western blot analysis of LC3-II and p62. Figure S5 (a) UCSC Genome Bioinformatics Site ( http://genome.ucsc.edu /) showed high enrichment of H3K27Ac at the promoter of MALAT1. (b) ChIP assays detected the H3K27Ac acetylation at promoter of MALAT1 in gastric cancer tissues. (c) ChIP assays detected the H3K27Ac acetylation at promoter of MALAT1 in gastric cancer cells.*, p < 0.05, **, p< 0.01. (d) The expression of the MALAT1 transcript (mean ± standard deviation) was detected using RT-PCR after cells were stimulated with varying concentrations of the histone deacetylase inhibitor trichostatin A (TSA) for 24 hr. Figure S6 (a) Compared with chemosensitive patients, LC3B and MALAT1 were markedly upregulated in chemoresistant patients using immunohistochemical analysis (for LC3B) and in situ hybridization analysis (for MALAT1). (b) According to data from The KMPlot database (TCGA), high MALAT1 expression resulted in a poorer disease-free survival (DFS, n=153, p=0.049) and overall survival (OS, n=153, p=0,039) in patients who received 5-Fu based adjuvant therapy. The HRs and p values were calculated with log-rank tests. Figure S7 (a) The copy number of MALAT1 or miR-23b-3p detected SGC7901/VCR cells, using RT-PCR and standard curves of known copy numbers of plasmid-derived reference standard. Error bars show standard deviation. (b) Cellular characterization of MALAT1 and miR-23b-3p, the levels of nuclear control transcript (U1), cytoplasmic control transcript (Actin mRNA), and MALAT1 were assessed by qRT-PCR in nuclear and cytoplasmic fractions in SGC7901/VCR cells. Data are presented as a percentage of U1, Actin and MALAT1 levels and total levels for each were taken to be 100%. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. Figure S8 (a) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant downregulation of miR-23b-3p as determined with qRT-PCR analysis. (b) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transduction of lentiviruses encoding miR-23b-3p mimic or control mimic. (c) SGC7901/VCR cells were transfected with sh-NC, sh-MALAT1, sh-MALAT1+miRNA-23b-3p inhibitor and miR-23b-3p inhibitor. qRT-PCR was performed 48 h post transfection. ATG12 and HMGB2 were determined with qRT-PCR analysis. Figure S9 (a) Compared with chemosensitive patients, ATG12 was markedly upregulated and miR-23b-3p was downregulated in chemoresistant patients using immunohistochemical analysis (for ATG12) and in situ hybridization analysis (for miR-23b-3p). (b,c) According to data from The KMPlot database (TCGA), low miR-23b-3p and high ATG12 expression resulted in a poorer disease-free survival and overall survival in patients who received 5-Fu based adjuvant therapy. The HRs and p values were calculated with log-rank tests. Table S1 Primer sequence used in this study. (DOCX 2642 kb)

Suppemental materials and methods. (DOCX 16 kb)

SUMMARY When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and � 10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1 Chk2 effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression—regressed leading or lagging strands—on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Whereas the actions of enhancers in gene transcriptional regulation are well established, roles of JmjC domain-containing proteins in mediating enhancer activation remain poorly understood. Here we report that recruitment of the JmjC domain-containing protein 6 (JMJD6) to estrogen receptor alpha (ERα)-bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers, resulting in transcriptional pause release of cognate estrogen target genes. JMJD6 was found to interact with MED12 in the mediator complex to regulate its recruitment. Unexpectedly, JMJD6 is necessary for MED12 to interact with CARM1, which methylates MED12 at multiple arginine sites and regulates its chromatin binding. Consistent with its role in transcriptional activation, JMJD6 is required for estrogen/ERα-induced breast cancer cell growth and tumorigenesis. Our data have uncovered a critical regulator of estrogen/ERα-induced enhancer, coding gene activation and breast cancer cell potency, providing a potential therapeutic target of ER-positive breast cancers.

Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.