R-Loop Functions in Brca1 -Associated Mammary Tumorigenesis.

Excessive R-loops, a DNA-RNA hybrid structure, are associated with genome instability and BRCA1 mutation-related breast cancer. Yet the causality of R-loops in tumorigenesis remains unclear. Here we show that R-loop removal by Rnaseh1 overexpression (Rh1-OE) in Brca1 -knockout (BKO) mouse mammary epithelium exacerbates DNA replication stress without affecting homology-directed DNA repair. R-loop removal also diminishes luminal progenitors, the cell of origin for estrogen receptor α (ERα)-negative BKO tumors. However, R-loop reduction does not dampen spontaneous BKO tumor incidence. Rather, it gives rise to a significant percentage of ERα-expressing BKO tumors. Thus, R-loops reshape mammary tumor subtype rather than promoting tumorigenesis.

An essential signaling function of cytoplasmic NELFB is independent of RNA polymerase II pausing.

The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits.

A novel mitochondria-related gene signature in esophageal carcinoma: prognostic, immune, and therapeutic features.

Esophageal carcinoma (ESCA) is a common and lethal malignant tumor worldwide. The mitochondrial biomarkers were useful in finding significant prognostic gene modules associated with ESCA owing to the role of mitochondria in tumorigenesis and progression. In the present work, we obtained the transcriptome expression profiles and corresponding clinical information of ESCA from The Cancer Genome Atlas (TCGA) database. Differential expressed genes (DEGs) were overlapped with 2030 mitochondria-related genes to get mitochondria-related DEGs. The univariate cox regression, Least Absolute Shrinkage and Selection Operator (LASSO) regression, and multivariate cox regression were sequentially used to define the risk scoring model for mitochondria-related DEGs, and its prognostic value was verified in the external datasets GSE53624. Based on the risk score, ESCA patients were divided into high- and low-risk groups. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to further investigate the difference between low- and high-risk groups at the gene pathway level. CIBERSORT was used to evaluate immune cell infiltration. The mutation difference between high- and low-risk groups was compared by using the R package "Maftools". Cellminer was used to assess the association between the risk scoring model and drug sensitivity. As the most important outcome of the study, a 6-gene risk scoring model (APOOL, HIGD1A, MAOB, BCAP31, SLC44A2, and CHPT1) was constructed from 306 mitochondria-related DEGs. Pathways including the "hippo signaling pathway" and "cell-cell junction" were enriched in the DEGs between high and low groups. According to CIBERSORT, samples with high-risk scores demonstrated a higher abundance of CD4+ T cells, NK cells, M0 and M2 macrophages, and a lower abundance of M1 macrophages. The immune cell marker genes were correlated with the risk score. In mutation analysis, the mutation rate of TP53 was significantly different between the high- and low-risk groups. Drugs with a strong correlation with the risk model were selected. In conclusion, we focused on the role of mitochondria-related genes in cancer development and proposed a prognostic signature for individualized integrative assessment.

Constructing a novel mitochondrial-related gene signature for evaluating the tumor immune microenvironment and predicting survival in stomach adenocarcinoma.

BACKGROUND: The incidence and mortality of gastric cancer ranks fifth and fourth worldwide among all malignancies, respectively. Accumulating evidences have revealed the close relationship between mitochondrial dysfunction and the initiation and progression of stomach cancer. However, rare prognostic models for mitochondrial-related gene risk have been built up in stomach cancer. METHODS: In current study, the expression and prognostic value of mitochondrial-related genes in stomach adenocarcinoma (STAD) patients were systematically analyzed to establish a mitochondrial-related risk model based on available TCGA and GEO databases. The tumor microenvironment (TME), immune cell infiltration, tumor mutation burden, and drug sensitivity of gastric adenocarcinoma patients were also investigated using R language, GraphPad Prism 8 and online databases. RESULTS: We established a mitochondrial-related risk prognostic model including NOX4, ALDH3A2, FKBP10 and MAOA and validated its predictive power. This risk model indicated that the immune cell infiltration in high-risk group was significantly different from that in the low-risk group. Besides, the risk score was closely related to TME signature genes and immune checkpoint molecules, suggesting that the immunosuppressive tumor microenvironment might lead to poor prognosis in high-risk groups. Moreover, TIDE analysis demonstrated that combined analysis of risk score and immune score, or stromal score, or microsatellite status could more effectively predict the benefit of immunotherapy in STAD patients with different stratifications. Finally, rapamycin, PD-0325901 and dasatinib were found to be more effective for patients in the high-risk group, whereas AZD7762, CEP-701 and methotrexate were predicted to be more effective for patients in the low-risk group. CONCLUSIONS: Our results suggest that the mitochondrial-related risk model could be a reliable prognostic biomarker for personalized treatment of STAD patients.

BRCA1 deficiency in mature CD8+ T lymphocytes impairs antitumor immunity.

Women with BRCA1 germline mutations have approximately an 80% lifetime chance of developing breast cancer. While the tumor suppressor function of BRCA1 in breast epithelium has been studied extensively, it is not clear whether BRCA1 deficiency in non-breast somatic cells also contribute to tumorigenesis. Here, we report that mouse Brca1 knockout (KO) in mature T lymphocytes compromises host antitumor immune response to transplanted syngeneic mouse mammary tumors. T cell adoptive transfer further corroborates CD8+ T cell-intrinsic impact of Brca1 KO on antitumor adaptive immunity. T cell-specific Brca1 KO mice exhibit fewer total CD8+, more exhausted, reduced cytotoxic, and reduced memory tumor-infiltrating T cell populations. Consistent with the preclinical data, cancer-free BRCA1 mutation-carrying women display lower abundance of circulating CD8+ lymphocytes than the age-matched control group. Thus, our findings support the notion that BRCA1 deficiency in adaptive immunity could contribute to BRCA1-related tumorigenesis. We also suggest that prophylactic boosting of adaptive immunity may reduce cancer incidence among at-risk women.

Phosphorylation of BRCA1 by ATM upon double-strand breaks impacts ATM function in end-resection: A potential feedback loop.

BRCA1 maintains genome stability by promoting homologous recombination (HR)-mediated DNA double-strand break (DSB) repair. Mutation of mouse BRCA1-S1152, corresponding to an ATM phosphorylation site in its human counterpart, resulted in increased genomic instability and tumor incidence. In this study, we report that BRCA1-S1152 is part of a feedback loop that sustains ATM activity. BRCA1-S1152A mutation impairs recruitment of the E3 ubiquitin ligase SKP2. This in turn attenuates NBS1-K63 ubiquitination by SKP2 at DSB, impairs sustained ATM activation, and ultimately leads to deficient end resection, the commitment step in the HR repair pathway. Auto-phosphorylation of human ATM at S1981 is known to be important for its kinase activation; we mutated the corresponding amino acid residue in mouse ATM (S1987A) to characterize potential roles of mouse ATM-S1987 in the BRCA1-SKP2-NBS1-ATM feedback loop. Unexpectedly, MEFs carrying the ATM-S1987A knockin mutation maintain damage-induced ATM kinase activation, suggesting a species-specific function of human ATM auto-phosphorylation.

RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased nonhomologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors sensitive to olaparib. In contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its cofactor BARD1 but instead coimmunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TANK-binding kinase 1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and immune-independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker. SIGNIFICANCE: PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion.

Immune exclusion predicts poor patient outcomes in multiple malignancies, including triple-negative breast cancer (TNBC)1. The extracellular matrix (ECM) contributes to immune exclusion2. However, strategies to reduce ECM abundance are largely ineffective or generate undesired outcomes3,4. Here we show that discoidin domain receptor 1 (DDR1), a collagen receptor with tyrosine kinase activity5, instigates immune exclusion by promoting collagen fibre alignment. Ablation of Ddr1 in tumours promotes the intratumoral penetration of T cells and obliterates tumour growth in mouse models of TNBC. Supporting this finding, in human TNBC the expression of DDR1 negatively correlates with the intratumoral abundance of anti-tumour T cells. The DDR1 extracellular domain (DDR1-ECD), but not its intracellular kinase domain, is required for immune exclusion. Membrane-untethered DDR1-ECD is sufficient to rescue the growth of Ddr1-knockout tumours in immunocompetent hosts. Mechanistically, the binding of DDR1-ECD to collagen enforces aligned collagen fibres and obstructs immune infiltration. ECD-neutralizing antibodies disrupt collagen fibre alignment, mitigate immune exclusion and inhibit tumour growth in immunocompetent hosts. Together, our findings identify a mechanism for immune exclusion and suggest an immunotherapeutic target for increasing immune accessibility through reconfiguration of the tumour ECM.

BRCA1 Antibodies Matter.

Breast cancer susceptibility gene 1 (BRCA1) encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes including DNA damage repair, cell cycle checkpoint activation, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation and apoptosis. A large number of BRCA1 antibodies have been generated and become commercially available over the past three decades, however, many commercial antibodies are poorly characterized and, when widely used, led to unreliable data. In search of reliable and specific BRCA1 antibodies (Abs), particularly antibodies recognizing mouse BRCA1, we performed a rigorous validation of a number of commercially available anti-BRCA1 antibodies, using proper controls in a panel of validation applications, including Western blot (WB), immunoprecipitation (IP), immunoprecipitation-mass spectrometry (IP-MS), chromatin immunoprecipitation (ChIP) and immunofluorescence (IF). Furthermore, we assessed the specificity of these antibodies to detect mouse BRCA1 protein through the use of testis tissue and mouse embryonic fibroblasts (MEFs) from Brca1+/+ and Brca1Δ11/Δ11 mice. We find that Ab1, D-9, 07-434 (for recognizing human BRCA1) and 287.17, 440621, BR-64 (for recognizing mouse BRCA1) are specific with high quality performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels.

A Phosphotyrosine Switch in Estrogen Receptor ß Is Required for Mouse Ovarian Function.

The two homologous estrogen receptors ERα and ERß exert distinct effects on their cognate tissues. Previous work from our laboratory identified an ERß-specific phosphotyrosine residue that regulates ERß transcriptional activity and antitumor function in breast cancer cells. To determine the physiological role of the ERß phosphotyrosine residue in normal tissue development and function, we investigated a mutant mouse model (Y55F) whereby this particular tyrosine residue in endogenous mouse ERß is mutated to phenylalanine. While grossly indistinguishable from their wild-type littermates, mutant female mice displayed reduced fertility, decreased ovarian follicular cell proliferation, and lower progesterone levels. Moreover, mutant ERß from female mice during superovulation is defective in activating promoters of its target genes in ovarian tissues. Thus, our findings provide compelling genetic and molecular evidence for a role of isotype-specific ERß phosphorylation in mouse ovarian development and function.

Estrogen receptor beta signaling in CD8+ T cells boosts T cell receptor activation and antitumor immunity through a phosphotyrosine switch.

BackgroundThe non-overlapping functions of the two estrogen receptor subtypes, ERα (Estrogen Receptor α)and ERß (Estrogen Receptor ß), in tumor cells have been studied extensively. However, their counterparts in host cells is vastly underinterrogated. Even less is known about how ERα and ERß activities are regulated in a subtype-specific manner. We previously identified a phosphotyrosine residue (pY36) of human ERß that is important for tumor ERß to inhibit growth of breast cancer cells in vitro and in vivo. A role of this ERß phosphotyrosine switch in regulating host ERß remains unclear.Conventional gene editing was used to mutate the corresponding tyrosine residue of endogenous mouse ERß (Y55F) in mouse embryonic stem cells. The derived homozygous mutant Esr2Y55F/Y55F mouse strain and its wild-type (WT) counterpart were compared in various transplant tumor models for their ability to support tumor growth. In addition, flow cytometry-based immunophenotyping was carried out to assess antitumor immunity of WT and mutant hosts. Adoptive transfer of bone marrow and purified CD8+ T cells were performed to identify the host cell type that harbors ERß-dependent antitumor function. Furthermore, cell signaling assays were conducted to compare T cell receptor (TCR)-initiated signaling cascade in CD8+ T cells of WT and mutant mice. Lastly, the ERß-selective agonist S-equol was evaluated for its efficacy to boost immune checkpoint blockade (ICB)-based anticancer immunotherapy.Disabling the ERß-specific phosphotyrosine switch in tumor-bearing hosts exacerbates tumor growth. Further, a cell-autonomous ERß function was defined in CD8+ effector T cells. Mechanistically, TCR activation triggers ERß phosphorylation, which in turn augments the downstream TCR signaling cascade via a non-genomic action of ERß. S-equol facilitates TCR activation that stimulates the ERß phosphotyrosine switch and boosts anti-PD-1 (Programmed cell death protein 1) ICB immunotherapy.Our mouse genetic study clearly demonstrates a role of the ERß phosphotyrosine switch in regulating ERß-dependent antitumor immunity in CD8+ T cells. Our findings support the development of ERß agonists including S-equol in combination with ICB immunotherapy for cancer treatment.

SRT2183 impairs ovarian cancer by facilitating autophagy.

The 5-year survival rate of ovarian cancer patients is only 47%, and developing novel drugs for ovarian cancer is needed. Herein, we evaluated if and how SRT2183, a sirtuin-1 activator, impairs the ovarian cancer cells. OVCAR-3 and A2780 cells were treated with SRT2183. Cell viability was measured by cell counting kit-8 assay and clonogenic assay. Apoptosis was determined by flow cytometry with Annexin V and propidium iodide. The level of autophagy was evaluated by western blot and immunofluorescence. The activities of AKT/mTOR/70s6k and MAPK signaling pathway were measured by immunoblot. SRT2183 inhibited the growth of ovarian cancer cells, increased the accumulation of BAX, cleaved-caspase 3 and cleaved-PARP, and decreased the level of anti-apoptotic Bcl-2 and Mcl-1. SRT2183 increased the LC3II level, and enhanced the degradation of p62/SQSTM1. SRT2183 increased the formation of GFP-LC3 puncta and induced the maturation of autophagosome. Interestingly, knockdown of autophagy related 5 and 7 significantly impaired the anti-carcinoma activity of SRT2183, implying that SRT2183 impaired the ovarian cancer cells by inducing autophagy. SRT2183 decreased the accumulation of p-Akt, p-mTOR and p-70s6k, and activated the p38 MAPK signaling pathway. This indicated that Akt/mTOR/70s6k and p38 MAPK signaling pathway might be involved in the SRT2183-mediated autophagy and apoptosis.

The 3D genomic landscape of differential response to EGFR/HER2 inhibition in endocrine-resistant breast cancer cells.

BACKGROUND: Recent studies suggested that crosstalk between ERα and EGFR/HER2 pathways plays a critical role in mediating endocrine therapy resistance. Several inhibitors targeting EGFR/HER2 signaling, including FDA-approved lapatinib and gefitinib as well as a novel dual tyrosine kinase inhibitor (TKI) sapitinib, showed greater therapeutic efficacies. However, how 3D chromatin landscape responds to the inhibition of EGFR/HER2 pathway remains to be elucidated. METHODS: In this study, we conducted in situ Hi-C and RNA-seq in two ERα+ breast cancer cell systems, 1) parental MCF7 cells and its associated tamoxifen-resistant MCF7TR cells; and 2) parental T47D cells and its associated tamoxifen-resistant T47DTR cells, before and after the treatment of sapitinib. RESULTS: We identified differential responses in topologically associated domains (TADs), looping genes and expressed genes. Interestingly, we found that many differential TADs and looping genes are reversible after sapitinib treatment, indicating that EGFR/HER2 signaling may play a role in reshaping and rewiring the high order genome organization. We further examined and recapitulated the reversible looping genes in 3D spheroids of breast cancer cells, demonstrating that 3D cell culture spheroid of breast cancer cells could be a potential preclinical breast cancer model for studying 3D chromatin regulation. CONCLUSIONS: Our study has provided significant insights into our understanding of 3D genomic landscape changes in response to EGFR/HER2 Inhibition in endocrine-resistant breast cancer cells. Our data provides a rich resource for further evaluating chromatin structural responses to EGFR/HER2 targeted therapies in endocrine-resistant breast cancer cells. Our analyses suggest that these alterations of chromatin structures and transcriptional programs may provide new avenues for intervention or designing of patient selection for targeted endocrine treatment.

Genetic ablation of adipocyte PD-L1 reduces tumor growth but accentuates obesity-associated inflammation.

The programmed death-ligand 1 (PD-L1)-dependent immune checkpoint attenuates host immunity and maintains self-tolerance. Imbalance between protective immunity and immunopathology due to altered PD-L1 signaling can lead to autoimmunity or tumor immunosuppression. The role of the PD-L1-dependent checkpoint in non-immune system is less reported. We previously found that white adipocytes highly express PD-L1. Here we show that adipocyte-specific PD-L1 knockout mice exhibit enhanced host anti-tumor immunity against mammary tumors and melanoma with low or no tumor PD-L1. However, adipocyte PD-L1 ablation in tumor-free mice also exacerbates diet-induced body weight gain, pro-inflammatory macrophage infiltration into adipose tissue, and insulin resistance. Low PD-L1 mRNA levels in human adipose tissue correlate with high body mass index and presence of type 2 diabetes. Therefore, our mouse genetic approach unequivocally demonstrates a cell-autonomous function of adipocyte PD-L1 in promoting tumor growth and inhibiting antitumor immunity. In addition, our work uncovers a previously unrecognized role of adipocyte PD-L1 in mitigating obesity-related inflammation and metabolic dysfunction.

Diabetes Distress in Young Adults with Type 2 Diabetes: A Cross-Sectional Survey in China.

BACKGROUND: Diabetes distress is a negative emotion related to diabetes management, which can compromise self-care and management of diabetes. However, few studies on diabetes distress have focused on young adults with type 2 diabetes in China. METHODS: A cross-sectional survey was conducted. Using a convenient sampling method, 98 young adults with type 2 diabetes who were admitted to our hospital from June 2017 to July 2018 were selected as research subjects. They were investigated using a basic demographic questionnaire, Diabetes Distress Scale, Summary of Diabetes Self-Care Activities Measure, and Audit of Disease Knowledge. Pearson's correlation analysis and regression analysis were used to analyze the influencing factors of diabetic distress. RESULTS: Among participants, 90.82% suffered from diabetes distress with an average score of 3.01 ± 0.58. Regimen-related, emotional burden-related, and interpersonal-related distress were the most frequently reported as severe. The results of the single-factor analysis showed that gender (P = 0.019), age (P = 0.003), occupation (P = 0.022), smoking (P < 0.001), and diabetes complications (P = 0.001) were the main factors affecting diabetes distress. The correlation analysis showed that diabetes distress was negatively correlated with the level of diabetic self-management (P < 0.001, r = -0.377) but not with the level of diabetes knowledge (P = 0.052, r = -0.197). The results of a multiple regression analysis showed that self-management level (P = 0.001, 95% CI: -0.039-0.011), age (P = 0.002, 95% CI: -0.463-0.104), smoking (P = 0.018, 95% CI: -0.504-0.048), and complications (P = 0.009, 95% CI: -0.517-0.076) accounted for 35.42% of the total variation in diabetes distress. CONCLUSION: Young adults with type 2 diabetes reported severe diabetes distress. Age, smoking, and diabetes complications were the main factors influencing diabetes distress in young adults with type 2 diabetes. Results of the present study are fundamental in selecting targeted measures for alleviating diabetes distress and thus improving the quality of life in these patients.

PPARγ inhibition boosts efficacy of PD-L1 Checkpoint Blockade Immunotherapy against Murine Melanoma in a sexually dimorphic manner.

Immune checkpoint blockade-based immunotherapy has become standard of care for multiple cancer types. However, the overall response rates among various cancer types still remain unsatisfactory. There is a pressing clinical need to identify combination therapies to improve efficacy of anticancer immunotherapy. We previously showed that pharmacologic inhibition of PPARγ by GW9662 boosts αPD-L1 and αPD-1 antibody efficacy in treating murine mammary tumors. In addition, we defined sexually dimorphic αPD-L1 efficacy in B16 melanoma. Here, we show a sexually dimorphic response to the combination of GW9662 and αPD-L1 immunotherapy in B16 melanoma. Combination effects were observed in female, but not male hosts. Neither female oöphorectomy impairs, nor does male castration rescue the combination effects, suggesting a sex hormone-independent response to this combination therapy. In diet-induced obese females, melanoma growth remained responsive to the combination treatment, albeit less robustly than lean females. These findings are informative for future design and application of immunotherapy-related combination therapy for treating human melanoma patients by taking gender and obesity status into consideration.

BRCA1 mutations attenuate super-enhancer function and chromatin looping in haploinsufficient human breast epithelial cells.

BACKGROUND: BRCA1-associated breast cancer originates from luminal progenitor cells. BRCA1 functions in multiple biological processes, including double-strand break repair, replication stress suppression, transcriptional regulation, and chromatin reorganization. While non-malignant cells carrying cancer-predisposing BRCA1 mutations exhibit increased genomic instability, it remains unclear whether BRCA1 haploinsufficiency affects transcription and chromatin dynamics in breast epithelial cells. METHODS: H3K27ac-associated super-enhancers were compared in primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Non-tumorigenic MCF10A breast epithelial cells with engineered BRCA1 haploinsufficiency were used to confirm the H3K27ac changes. The impact of BRCA1 mutations on enhancer function and enhancer-promoter looping was assessed in MCF10A cells. RESULTS: Here, we show that primary mammary epithelial cells from women with BRCA1 mutations display significant loss of H3K27ac-associated super-enhancers. These BRCA1-dependent super-enhancers are enriched with binding motifs for the GATA family. Non-tumorigenic BRCA1mut/+ MCF10A cells recapitulate the H3K27ac loss. Attenuated histone mark and enhancer activity in these BRCA1mut/+ MCF10A cells can be partially restored with wild-type BRCA1. Furthermore, chromatin conformation analysis demonstrates impaired enhancer-promoter looping in BRCA1mut/+ MCF10A cells. CONCLUSIONS: H3K27ac-associated super-enhancer loss is a previously unappreciated functional deficiency in ostensibly normal BRCA1 mutation-carrying breast epithelium. Our findings offer new mechanistic insights into BRCA1 mutation-associated transcriptional and epigenetic abnormality in breast epithelial cells and tissue/cell lineage-specific tumorigenesis.

BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells.

BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.

Shenqi Fuzheng Injection impairs bile duct ligation-induced cholestatic liver injury in vivo.

BACKGROUND AND AIM: The aim of the present study sought to determine the protective function of Shenqi Fuzheng Injection (SFI) in cholestatic liver injury. METHODS: Cholestatic liver injury was induced in a 7-day bile duct-ligated (BDL) rat model. Rats were divided into three groups that were comprised of: (1) Sham; (2) BDL model; and (3) SFI treatment. The sham and BDL groups were treated with an appropriate volume of 0.9% sodium chloride as the vehicle, and the SFI group was administered SFI at a dose of 20 ml/kg/day, via tail vein injection. RESULTS: SFI significantly (all at P<0.01) decreased the levels of serum aspartate aminotransferase and alanine aminotransferase as compared with the BDL group, which was associated with reduced severity of inflammatory cell infiltration and hepatic damage. Moreover, SFI significantly decreased the levels of hepatic interleukin-6 (P<0.01), tumor necrosis factor-α (P=0.041), and malondialdehyde (P=0.026), and significantly increased the levels of total superoxide dismutase (P<0.01), and the GSH/GSSG ratio (P=0.041) in the liver. Western blot analysis showed that SFI increased PPAR-γ expression; however, SFI treatment decreased cyclooxygenase-2 (COX-2) expression and the phosphorylation of NF-κBp65. CONCLUSIONS: These data demonstrated that SFI attenuated both inflammation and oxidative stress, and disrupted cholestatic liver injury. The involved mechanism was dependent, at least in part, on regulating PPAR-γ, COX-2, and NF-κBp65 expression.