Detection and characterization of pancreatic and biliary tract cancers using cell-free DNA fragmentomics.

BACKGROUND: Plasma cell-free DNA (cfDNA) fragmentomics has demonstrated significant differentiation power between cancer patients and healthy individuals, but little is known in pancreatic and biliary tract cancers. The aim of this study is to characterize the cfDNA fragmentomics in biliopancreatic cancers and develop an accurate method for cancer detection. METHODS: One hundred forty-seven patients with biliopancreatic cancers and 71 non-cancer volunteers were enrolled, including 55 patients with cholangiocarcinoma, 30 with gallbladder cancer, and 62 with pancreatic cancer. Low-coverage whole-genome sequencing (median coverage: 2.9 ×) was performed on plasma cfDNA. Three cfDNA fragmentomic features, including fragment size, end motif and nucleosome footprint, were subjected to construct a stacked machine learning model for cancer detection. Integration of carbohydrate antigen 19-9 (CA19-9) was explored to improve model performance. RESULTS: The stacked model presented robust performance for cancer detection (area under curve (AUC) of 0.978 in the training cohort, and AUC of 0.941 in the validation cohort), and remained consistent even when using extremely low-coverage sequencing depth of 0.5 × (AUC: 0.905). Besides, our method could also help differentiate biliopancreatic cancer subtypes. By integrating the stacked model and CA19-9 to generate the final detection model, a high accuracy in distinguishing biliopancreatic cancers from non-cancer samples with an AUC of 0.995 was achieved. CONCLUSIONS: Our model demonstrated ultrasensitivity of plasma cfDNA fragementomics in detecting biliopancreatic cancers, fulfilling the unmet accuracy of widely-used serum biomarker CA19-9, and provided an affordable way for accurate noninvasive biliopancreatic cancer screening in clinical practice.

Genomic and transcriptomic characteristics of 12 novel primary cell lines derived from three patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive bile duct malignancy with poor prognosis. To improve our understanding of the biological characteristics of CCA and develop effective therapies, appropriate preclinical models are required. Here, we established and characterized 12 novel patient-derived primary cancer cell (PDPC) models using multi-region sampling. At the genomic level of PDPCs, we observed not only commonly mutated genes, such as TP53, JAK3, and KMT2C, consistent with the reports in CCA, but also specific mutation patterns in each cell line. In addition, specific expression patterns with distinct biological functions and pathways involved were also observed in the PDPCs at the transcriptomic level. Furthermore, the drug-sensitivity results revealed that the PDPCs exhibited different responses to the six commonly used compounds. Our findings indicate that the established PDPCs can serve as novel in vitro reliable models to provide a crucial molecular basis for improving the understanding of tumorigenesis and its treatment.

Single-cell transcriptomics reveal the heterogeneity and dynamic of cancer stem-like cells during breast tumor progression.

Breast cancer stem-like cells (BCSCs) play vital roles in tumorigenesis and progression. However, the origin and dynamic changes of BCSCs are still to be elucidated. Using the breast cancer mouse model MMTV-PyMT, we constructed a single-cell atlas of 31,778 cells from four distinct stages of tumor progression (hyperplasia, adenoma/MIN, early carcinoma and late carcinoma), during which malignant transition occurs. We identified that the precise cell type of ERlow epithelial cell lineage gave rise to the tumors, and the differentiation of ERhigh epithelial cell lineage was blocked. Furthermore, we discovered a specific signature with a continuum of gene expression profiles along the tumor progression and significantly correlated with clinical outcomes, and we also found a stem-like cell cluster existed among ERlow epithelial cells. Further clustering on this stem-like cluster showed several sub-clusters indicating heterogeneity of stem-like epithelial cells. Moreover, we distinguished normal and cancer stem-like cells in this stem-like epithelial cell cluster and profiled the molecular portraits from normal stem-like cell to cancer stem-like cells during the malignant transition. Finally, we found the diverse immune cell infiltration displayed immunosuppressive characteristics along tumor progression. We also found the specific expression pattern of cytokines and their corresponding cytokine receptors in BCSCs and immune cells, suggesting the possible cross-talk between BCSCs and the immune cells. These data provide a useful resource for illuminating BCSC heterogeneity and the immune cell remodeling during breast tumor progression, and shed new light on transcriptomic dynamics during the progression at the single-cell level.

TEM8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer.

Enhanced neovasculogenesis, especially vasculogenic mimicry (VM), contributes to the development of triple-negative breast cancer (TNBC). Breast tumor-initiating cells (BTICs) are involved in forming VM; however, the specific VM-forming BTIC population and the regulatory mechanisms remain undefined. We find that tumor endothelial marker 8 (TEM8) is abundantly expressed in TNBC and serves as a marker for VM-forming BTICs. Mechanistically, TEM8 increases active RhoC level and induces ROCK1-mediated phosphorylation of SMAD5, in a cascade essential for promoting stemness and VM capacity of breast cancer cells. ASB10, an estrogen receptor ERα trans-activated E3 ligase, ubiquitylates TEM8 for degradation, and its deficiency in TNBC resulted in a high homeostatic level of TEM8. In this work, we identify TEM8 as a functional marker for VM-forming BTICs in TNBC, providing a target for the development of effective therapies against TNBC targeting both BTIC self-renewal and neovasculogenesis simultaneously.

Interfering MSN-NONO complex-activated CREB signaling serves as a therapeutic strategy for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is life-threatening because of limited therapies and lack of effective therapeutic targets. Here, we found that moesin (MSN) was significantly overexpressed in TNBC compared with other subtypes of breast cancer and was positively correlated with poor overall survival. However, little is known about the regulatory mechanisms of MSN in TNBC. We found that MSN significantly stimulated breast cancer cell proliferation and invasion in vitro and tumor growth in vivo, requiring the phosphorylation of MSN and a nucleoprotein NONO-assisted nuclear localization of phosphorylated MSN with protein kinase C (PKC) and then the phosphorylation activation of CREB signaling by PKC. Our study also demonstrated that targeting MSN, NONO, or CREB significantly inhibited breast tumor growth in vivo. These results introduce a new understanding of MSN function in breast cancer and provide favorable evidence that MSN or its downstream molecules might serve as new targets for TNBC treatment.

General mechanism of JQ1 in inhibiting various types of cancer.

Bromodomain­containing 4 (BRD4) is a histone modification reader and transcriptional regulator that has been reported to interact with acetylated lysine histone motifs transcription factors (TFs), transcription co­activators and RNA polymerase II. The selective small molecule inhibitor JQ1, which binds competitively to bromodomains, has been reported to exhibit anti­proliferative effects in various types of cancer. Previous studies on the mechanism of action of JQ1 mostly focused on a specific tumor type or disease; however, the general mechanism through which JQ1 affects various tumors remains to be determined. In the present study, chromatin immunoprecipitation sequencing data for BRD4 and its expression profiles in six cancer cell lines were integrated and analyzed systematically. The results indicated that BRD4 binds to enhancers with histone H3 acetylated at lysine 27 (H3K27Ac) and mediator complex subunit 1 in a cell type­specific manner, as well as binds to promoter regions with the oncogenic TFs MYC and E2F1 in a cell type­common manner. The cell type­common sites across the six cell types investigated were found to be functionally important for tumorigenesis, whereas the cell type­specific sites were functionally enriched with the cell identity, all of which were sensitive to JQ1 treatment. Furthermore, a core set of JQ1­regulated BRD4 binding genes were obtained, which were significantly inhibited by JQ1 in various cancer cell lines and contributed to hallmarks of cancer. These results implied a common mechanism underlying the therapeutic effects of JQ1 and suggested its potential suitability as an anti­cancer drug targeting BRD4­mediated transcriptional regulation.

KDM3B suppresses APL progression by restricting chromatin accessibility and facilitating the ATRA-mediated degradation of PML/RARα.

BACKGROUND: A hallmark of acute promyelocytic leukemia (APL) is the expression of PML/RARα fusion protein. Treatment with all-trans retinoic acid (ATRA) results in the terminal differentiation of neutrophil granulocytes. However, the underlying mechanisms remain largely unknown. Here, we identify and elucidate a novel differentiation-suppressive model of APL involving the histone demethylase KDM3B, which has been identified as a suppressor of the tumor genes involved in hematopoietic malignancies. METHODS: First, we established a KDM3B knockdown NB4 cell model to determine the functional characteristics of KDM3B by cell proliferation assay and flow cytometry. Then, we performed ChIP-seq and ATAC-seq to search for potential relationships among KDM3B, histone modification (H3K9me1/me2) and the chromatin state. Finally, molecular biological techniques and a multi-omics analysis were used to explore the role of KDM3B in differentiation of the leukemia cells after ATRA treatment. RESULTS: We found that knocking down KDM3B contributed to the growth of NB4 APL cells via the promotion of cell-cycle progression and blocked granulocytic differentiation. Through global and molecular approaches, we provided futher evidence that knocking down KDM3B altered the global distribution of H3K9me1/me2 and increased the chromatin accessibility. Moreover, knocking down KDM3B inhibited the ATRA-induced degradation of the PML/RARα oncoprotein. CONCLUSION: Our study suggested that KDM3B was able to inhibit APL progression by maintaining chromatin in a compact state and facilitating the ATRA-mediated degradation of PML/RARα. Taken together, the results show that KDM3B may be an alternative target for the treatment regimens and the targeted therapy for APL by sustaining the function of PML/RARα fusion protein.

Cooperativity of co-factor NR2F2 with Pioneer Factors GATA3, FOXA1 in promoting ERα function.

Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically. Methods: Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information (RNA-seq, ATAC-seq, Gene microarray, 450k methylation chip, GRO-seq), forming a multi-dimension network to illuminate ERα associated transcription. Results: We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers. Conclusions: The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors (NR2F2, FOXA1 and GATA3).The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription.

Synergy between arsenic trioxide and JQ1 on autophagy in pancreatic cancer.

Pancreatic cancer is a deadliest type of malignancy and lacks effective intervention. We here report a potential strategy for treatment of this malignancy by the combination of arsenic trioxide (ATO) and BET bromodomain inhibitor JQ1. These two agents synergistically modulate multistages of autophagy and thus induce apoptosis effectively in pancreatic cancer cells. Our genomic and biochemical data have demonstrated that crosstalks between ER stress and autophagy play crucial roles during ATO-induced apoptosis, in which NRF2 may stand at the crossroad between cell death and survival. This has been further strengthened by our finding that NRF2 depletion renders insensitive cells into sensitive ones in regard to ATO treatment-caused cell death. The knockdown of NRF2 and the addition of JQ1 result in similar molecular/cellular effects in promoting effective ATO-induced apoptosis in cells that are insensitive to ATO treatment alone. Thus, the combination of ATO and JQ1 may represent a new treatment strategy for pancreatic cancer.