Cancer-specific epigenome identifies oncogenic hijacking by nuclear factor I family proteins for medulloblastoma progression.

Normal cells coordinate proliferation and differentiation by precise tuning of gene expression based on the dynamic shifts of the epigenome throughout the developmental timeline. Although non-mutational epigenetic reprogramming is an emerging hallmark of cancer, the epigenomic shifts that occur during the transition from normal to malignant cells remain elusive. Here, we capture the epigenomic changes that occur during tumorigenesis in a prototypic embryonal brain tumor, medulloblastoma. By comparing the epigenomes of the different stages of transforming cells in mice, we identify nuclear factor I family of transcription factors, known to be cell fate determinants in development, as oncogenic regulators in the epigenomes of precancerous and cancerous cells. Furthermore, genetic and pharmacological inhibition of NFIB validated a crucial role of this transcription factor by disrupting the cancer epigenome in medulloblastoma. Thus, this study exemplifies how epigenomic changes contribute to tumorigenesis via non-mutational mechanisms involving developmental transcription factors.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Diffuse large B-cell lymphoma with DNA copy number changes in a Japanese black calf.

A 2-month-old Japanese Black calf exhibited mandibular and superficial cervical lymph node swelling. Fine needle aspiration cytology of the superficial cervical lymph node revealed large lymphoblast-like cells with mitoses. Hematological examination revealed remarkable lymphocytosis with atypical lymphocytes. Increased activities of serum total lactate dehydrogenase and thymidine kinase were detected. At necropsy, generalized swelling of lymph nodes was observed. Histopathological analysis revealed diffuse proliferation of medium-sized round centroblastic neoplastic cells that were positive for CD20, CD79α, PAX5, and BLA-36, and negative for CD3, CD5, CD10, and CD34. The calf was diagnosed with centroblastic diffuse large B-cell lymphoma (DLBCL) based on these findings. Analysis of DNA copy number variation revealed an increased copy number for the GIMAP family relative to that in healthy cattle. Moreover, decreases in copy numbers of GBP-1, MIR3141, OR5P1E, and PTPRG relative to those in healthy cattle were also observed. Because DNA copy number variation represent a major contribution to the somatic mutation landscapes in human tumors, these findings suggest that DNA copy number changes might have contributed to the onset of DLBCL in the present case.

Identification of lineage-specific epigenetic regulators FOXA1 and GRHL2 through chromatin accessibility profiling in breast cancer cell lines.

Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2-), 3 ER+/HER2+, 3 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P-specific regions while suppressing Group-B-specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.

Inhibition of protein arginine methyltransferase 6 activates interferon signaling and induces the apoptosis of endometrial cancer cells via histone modification.

Histone modification, a major epigenetic mechanism regulating gene expression through chromatin remodeling, introduces dynamic changes in chromatin architecture. Protein arginine methyltransferase 6 (PRMT6) is overexpressed in various types of cancer, including prostate, lung and endometrial cancer (EC). Epigenome regulates the expression of endogenous retrovirus (ERV), which activates interferon signaling related to cancer. The antitumor effects of PRMT6 inhibition and the role of PRMT6 in EC were investigated, using epigenome multi­omics analysis, including an assay for chromatin immunoprecipitation sequencing (ChIP­seq) and RNA sequencing (RNA­seq). The expression of PRMT6 in EC was analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and immunohistochemistry (IHC). The prognostic impact of PRMT6 expression was evaluated using IHC. The effects of PRMT6­knockdown (KD) were investigated using cell viability and apoptosis assays, as well as its effects on the epigenome, using ChIP­seq of H3K27ac antibodies and RNA­seq. Finally, the downstream targets identified by multi­omics analysis were evaluated. PRMT6 was overexpressed in EC and associated with a poor prognosis. PRMT6­KD induced histone hypomethylation, while suppressing cell growth and apoptosis. ChIP­seq revealed that PRMT6 regulated genomic regions related to interferons and apoptosis through histone modifications. The RNA­seq data demonstrated altered interferon­related pathways and increased expression of tumor suppressor genes, including NK6 homeobox 1 and phosphoinositide­3­kinase regulatory subunit 1, following PRMT6­KD. RT­qPCR revealed that eight ERV genes which activated interferon signaling were upregulated by PRMT6­KD. The data of the present study suggested that PRMT6 inhibition induced apoptosis through interferon signaling activated by ERV. PRMT6 regulated tumor suppressor genes and may be a novel therapeutic target, to the best of our knowledge, in EC.

Targeting DHX9 Triggers Tumor-Intrinsic Interferon Response and Replication Stress in Small Cell Lung Cancer.

Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as "viral mimicry," has emerged as an effective strategy to convert immunologically "cold" tumors into "hot." Through a curated CRISPR-based screen of RNA helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLC). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune-checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other "cold" tumors in which genomic instability contributes to pathology. SIGNIFICANCE: One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing endogenous "virus-mimetic" nucleic acid accumulation. Here, we identify DHX9 as a viral-mimicry-inducing factor involved in the suppression of double-stranded RNAs and R-loops and propose DHX9 as a novel target to enhance antitumor immunity. See related commentary by Chiappinelli, p. 389. This article is featured in Selected Articles from This Issue, p. 384.

Glutamine deficiency drives transforming growth factor-ß signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-ß (TGF-ß)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-ß-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-ß-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-ß-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-ß-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-ß signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Increased RUNX3 expression mediates tumor-promoting ability of human breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major stromal component of human breast cancers and often promote tumor proliferation, progression and malignancy. We previously established an experimental CAF (exp-CAF) cell line equipped with a potent tumor-promoting ability. It was generated through prolonged incubation of immortalized human mammary fibroblasts with human breast cancer cells in a tumor xenograft mouse model. RESULTS: Herein, we found that the exp-CAFs highly express Runt-related transcription factor 3 (RUNX3), while counterpart fibroblasts do not. In breast cancer patients, the proportion of RUNX3-positive stromal fibroblast-like cells tends to be higher in cancerous regions than in non-cancerous regions. These findings suggest an association of RUNX3 with CAF characteristics in human breast cancers. To investigate the functional role of RUNX3 in CAFs, the exp-CAFs with or without shRNA-directed knockdown of RUNX3 were implanted with breast cancer cells subcutaneously in immunodeficient mice. Comparison of the resulting xenograft tumors revealed that tumor growth was significantly attenuated when RUNX3 expression was suppressed in the fibroblasts. Consistently, Ki-67 and CD31 immunohistochemical staining of the tumor sections indicated reduction of cancer cell proliferation and microvessel formation in the tumors formed with the RUNX3-suppressed exp-CAFs. CONCLUSION: These results suggest that increased RUNX3 expression could contribute to the tumor-promoting ability of CAFs through mediating cancer cell growth and neoangiogenesis in human breast tumors.

TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells.

Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation.

Chromatin conformational changes at human satellite II contribute to the senescence phenotype in the tumor microenvironment.

Cellular senescence and senescence-associated secretory phenotype (SASP) in stromal cells within the tumor microenvironment promote cancer progression. Although cellular senescence has been shown to induce changes in the higher-order chromatin structure and abnormal transcription of repetitive elements in the genome, the functional significance of these changes is unclear. In this study, we examined the human satellite II (hSATII) loci in the pericentromere to understand these changes and their functional significance. Our results indicated that the hSATII loci decompact during senescence induction, resulting in new DNA-DNA interactions in distinct genomic regions, which we refer to as DRISR (Distinctive Regions Interacted with Satellite II in Replicative senescent Fibroblasts). Interestingly, decompaction occurs before the expression of hSATII RNA. The DRISR with altered chromatin accessibility was enriched for motifs associated with cellular senescence and inflammatory SASP genes. Moreover, DNA-fluorescence in situ hybridization analysis of the breast cancer tissues revealed hSATII decompaction in cancer and stromal cells. Furthermore, we reanalyzed the single-cell assay for transposase-accessible chromatin with sequencing data and found increased SASP-related gene expression in fibroblasts exhibiting hSATII decompaction in breast cancer tissues. These findings suggest that changes in the higher-order chromatin structure of the pericentromeric repetitive sequences during cellular senescence might directly contribute to the cellular senescence phenotype and cancer progression via inflammatory gene expression.

FOXD1 is associated with poor outcome and maintains tumor-promoting enhancer-gene programs in basal-like breast cancer.

Breast cancer biology varies markedly among patients. Basal-like breast cancer is one of the most challenging subtypes to treat because it lacks effective therapeutic targets. Despite numerous studies on potential targetable molecules in this subtype, few targets have shown promise. However, the present study revealed that FOXD1, a transcription factor that functions in both normal development and malignancy, is associated with poor prognosis in basal-like breast cancer. We analyzed publicly available RNA sequencing data and conducted FOXD1-knockdown experiments, finding that FOXD1 maintains gene expression programs that contribute to tumor progression. We first conducted survival analysis of patients grouped via a Gaussian mixture model based on gene expression in basal-like tumors, finding that FOXD1 is a prognostic factor specific to this subtype. Then, our RNA sequencing and chromatin immunoprecipitation sequencing experiments using the basal-like breast cancer cell lines BT549 and Hs578T with FOXD1 knockdown revealed that FOXD1 regulates enhancer-gene programs related to tumor progression. These findings suggest that FOXD1 plays an important role in basal-like breast cancer progression and may represent a promising therapeutic target.

HEY1-NCOA2 expression modulates chondrogenic differentiation and induces mesenchymal chondrosarcoma in mice.

Mesenchymal chondrosarcoma affects adolescents and young adults, and most cases usually have the HEY1::NCOA2 fusion gene. However, the functional role of HEY1-NCOA2 in the development and progression of mesenchymal chondrosarcoma remains largely unknown. This study aimed to clarify the functional role of HEY1-NCOA2 in transformation of the cell of origin and induction of typical biphasic morphology of mesenchymal chondrosarcoma. We generated a mouse model for mesenchymal chondrosarcoma by introducing HEY1-NCOA2 into mouse embryonic superficial zone (eSZ) followed by subcutaneous transplantation into nude mice. HEY1-NCOA2 expression in eSZ cells successfully induced subcutaneous tumors in 68.9% of recipients, showing biphasic morphologies and expression of Sox9, a master regulator of chondrogenic differentiation. ChIP sequencing analyses indicated frequent interaction between HEY1-NCOA2 binding peaks and active enhancers. Runx2, which is important for differentiation and proliferation of the chondrocytic lineage, is invariably expressed in mouse mesenchymal chondrosarcoma, and interaction between HEY1-NCOA2 and Runx2 is observed using NCOA2 C-terminal domains. Although Runx2 knockout resulted in significant delay in tumor onset, it also induced aggressive growth of immature small round cells. Runx3, which is also expressed in mesenchymal chondrosarcoma and interacts with HEY1-NCOA2, replaced the DNA-binding property of Runx2 only in part. Treatment with the HDAC inhibitor panobinostat suppressed tumor growth both in vitro and in vivo, abrogating expression of genes downstream of HEY1-NCOA2 and Runx2. In conclusion, HEY1::NCOA2 expression modulates the transcriptional program in chondrogenic differentiation, affecting cartilage-specific transcription factor functions.

ASPSCR1::TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a soft part malignancy affecting adolescents and young adults. ASPS is characterized by a highly integrated vascular network, and its high metastatic potential indicates the importance of ASPS's prominent angiogenic activity. Here, we find that the expression of ASPSCR1::TFE3, the fusion transcription factor causatively associated with ASPS, is dispensable for in vitro tumor maintenance; however, it is required for in vivo tumor development via angiogenesis. ASPSCR1::TFE3 is frequently associated with super-enhancers (SEs) upon its DNA binding, and the loss of its expression induces SE-distribution dynamic modification related to genes belonging to the angiogenesis pathway. Using epigenomic CRISPR/dCas9 screening, we identify Pdgfb, Rab27a, Sytl2, and Vwf as critical targets associated with reduced enhancer activities due to the ASPSCR1::TFE3 loss. Upregulation of Rab27a and Sytl2 promotes angiogenic factor-trafficking to facilitate ASPS vascular network construction. ASPSCR1::TFE3 thus orchestrates higher ordered angiogenesis via modulating the SE activity.

Chromatin profile-based identification of a novel ER-positive breast cancer subgroup with reduced ER-responsive element accessibility.

BACKGROUND: Oestrogen receptor (ER) signalling-dependent cancer cell growth is one of the major features of ER-positive breast cancer (BC). Inhibition of ER function is a standard and effective treatment for ER-positive tumours; however, ~20% of patients with ER-positive BC experience early or late recurrence. In this study, we examined intertumour heterogeneity from an epigenetic perspective based on the hypothesis that the intrinsic difference in epigenetic states around ER signalling pathway underlies endocrine therapy resistance. METHODS: We performed transposase-accessible chromatin sequencing (ATAC-seq) analysis of 42 BC samples, including 35 ER-positive(+) human epidermal growth factor receptor 2 (HER2)-negative(-) and 7 triple-negative tumours. We also reanalysed ATAC-seq data of 45 ER + /HER2 - tumours in the Cancer Genome Atlas (TCGA) BC cohort to validate our observations. RESULTS: We conducted a comprehensive analysis of cis-regulatory elements (CREs) using ATAC-seq, identifying three subgroups based on chromatin accessibility profiles. We identified a subgroup of ER-positive BCs with a distinctive chromatin accessibility pattern including reduced accessibility to ER-responsive elements (EREs). The same subgroup was also observed in TCGA BC cohort. Despite the reduced accessibility to EREs, the expression of ER and potential ER target genes were not decreased in these tumours. CONCLUSION: Our findings highlight the existence of a subset of ER-positive BCs with unchanged ER expression but reduced EREs accessibility that cannot be distinguished by conventional immunostaining for ER. Future studies should determine whether these tumours are associated with resistance to endocrine therapy.

Transcriptomic intratumor heterogeneity of breast cancer patient-derived organoids may reflect the unique biological features of the tumor of origin.

BACKGROUND: The intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs. METHODS: We established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO. RESULTS: Cancer cells were clustered into 3-6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial-mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients. CONCLUSIONS: We confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.

Two target gene activation pathways for orphan ERR nuclear receptors.

Estrogen-related receptors (ERRα/ß/γ) are orphan nuclear receptors that function in energy-demanding physiological processes, as well as in development and stem cell maintenance, but mechanisms underlying target gene activation by ERRs are largely unknown. Here, reconstituted biochemical assays that manifest ERR-dependent transcription have revealed two complementary mechanisms. On DNA templates, ERRs activate transcription with just the normal complement of general initiation factors through an interaction of the ERR DNA-binding domain with the p52 subunit of initiation factor TFIIH. On chromatin templates, activation by ERRs is dependent on AF2 domain interactions with the cell-specific coactivator PGC-1α, which in turn recruits the ubiquitous p300 and MED1/Mediator coactivators. This role of PGC-1α may also be fulfilled by other AF2-interacting coactivators like NCOA3, which is shown to recruit Mediator selectively to ERRß and ERRγ. Importantly, combined genetic and RNA-seq analyses establish that both the TFIIH and the AF2 interaction-dependent pathways are essential for ERRß/γ-selective gene expression and pluripotency maintenance in embryonic stem cells in which NCOA3 is a critical coactivator.

Estrogen receptor α K303R mutation reorganizes its binding to forkhead box protein A1 regions and induces chromatin opening.

BACKGROUND: Estrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown. METHODS AND RESULTS: We introduced the K303R ERα mutant in ERα-negative MDA-MB-453 cells to monitor ERα-dependent transactivation and to perform epigenomic analyses. ATAC-seq and ChIP-Seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Forkhead box (Fox) protein family motif regions at similar rates, even without an ERα-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant can be tethered on DNA by FoxA1 indirectly, but only the K303R/FoxA1/DNA complex can induce associations with the nuclear receptor cofactor 2 (NCOA2). CONCLUSIONS: These findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through the FoxA1-dependent associations of the K303R mutant to NCOA2 and then probably disrupts the regulation of Fox-target genes, resulting in K303R-related BC events.

GRHL2 motif is associated with intratumor heterogeneity of cis-regulatory elements in luminal breast cancer.

In breast cancer patients, tumor heterogeneity is associated with prognosis and therapeutic response; however, the epigenetic diversity that exists in primary tumors remains unknown. Using a single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), we obtained the chromatin accessibility profiles of 12,452 cells from 16 breast cancer patients including 11 luminal, 1 luminal-HER2, 1 HER2+, and 3 triple-negative subtypes. Via this profiling process, tumors were classified into cancer cells and the tumor microenvironment, highlighting the heterogeneity of disease-related pathways including estrogen receptor (ER) signaling. Furthermore, the coexistence of cancer cell clusters with different ER binding motif enrichments was identified in a single ER+ tumor. In a cluster with reduced ER motif enrichment, we identified GRHL2, a transcription factor, as the most enriched motif, and it cooperated with FOXA1 to initiate endocrine resistance. Coaccessibility analysis revealed that GRHL2 binding elements potentially regulate genes associated with endocrine resistance, metastasis, and poor prognosis in patients that received hormonal therapy. Overall, our study suggests that epigenetic heterogeneity could lead to endocrine resistance and poor prognosis in breast cancer patients and it offers a large-scale resource for further cancer research.

The oncogene-dependent resistance to reprogramming unveils cancer therapeutic targets.

The resistance to transcription factor-mediated reprogramming into pluripotent stem cells is one of the distinctive features of cancer cells. Here we dissect the profiles of reprogramming factor binding and the subsequent transcriptional response in cancer cells to reveal its underlying mechanisms. Using clear cell sarcomas (CCSs), we show that the driver oncogene EWS/ATF1 misdirects the reprogramming factors to cancer-specific enhancers and thereby impairs the transcriptional response toward pluripotency that is otherwise provoked. Sensitization to the reprogramming cue is observed in other cancer types when the corresponding oncogenic signals are pharmacologically inhibited. Exploiting this oncogene dependence of the transcriptional "stiffness," we identify mTOR signaling pathways downstream of EWS/ATF1 and discover that inhibiting mTOR activity substantially attenuates the propagation of CCS cells in vitro and in vivo. Our results demonstrate that the early transcriptional response to cell fate perturbations can be a faithful readout to identify effective therapeutics targets in cancer cells.