QDPR deficiency drives immune suppression in pancreatic cancer.

The relevance of biopterin metabolism in resistance to immune checkpoint blockade (ICB) therapy remains unknown. We demonstrate that the deficiency of quinoid dihydropteridine reductase (QDPR), a critical enzyme regulating biopterin metabolism, causes metabolite dihydrobiopterin (BH2) accumulation and decreases the ratio of tetrahydrobiopterin (BH4) to BH2 in pancreatic ductal adenocarcinomas (PDACs). The reduced BH4/BH2 ratio leads to an increase in reactive oxygen species (ROS) generation and a decrease in the distribution of H3K27me3 at CXCL1 promoter. Consequently, myeloid-derived suppressor cells are recruited to tumor microenvironment via CXCR2 causing resistance to ICB therapy. We discovered that BH4 supplementation is capable to restore the BH4/BH2 ratio, enhance anti-tumor immunity, and overcome ICB resistance in QDPR-deficient PDACs. Tumors with lower QDPR expression show decreased responsiveness to ICB therapy. These findings offer a novel strategy for selecting patient and combining therapies to improve the effectiveness of ICB therapy in PDAC.

Single-cell transcriptomic analysis deciphers heterogenous cancer stem-like cells in colorectal cancer and their organ-specific metastasis.

OBJECTIVE: Metastasis is the major cause of cancer death. However, what types of heterogenous cancer cells in primary tumour and how they metastasise to the target organs remain largely undiscovered. DESIGN: We performed single-cell RNA sequencing and spatial transcriptomic analysis in primary colorectal cancer (CRC) and metastases in the liver (lCRC) or ovary (oCRC). We also conducted immunofluorescence staining and functional experiments to examine the mechanism. RESULTS: Integrative analyses of epithelial cells reveal a stem-like cell cluster with high protein tyrosine phosphatase receptor type O (PTPRO) and achaete scute-like 2 (ASCL2) expression as the metastatic culprit. This cell cluster comprising distinct subpopulations shows distinct liver or ovary metastatic preference. Population 1 (P1) cells with high delta-like ligand 4 (DLL4) and MAF bZIP transcription factor A (MAFA) expression are enriched in primary CRC and oCRC, thus may be associated with ovarian metastasis. P3 cells having a similar expression pattern as cholangiocytes are found mainly in primary CRC and lCRC, presuming to be likely the culprits that specifically metastasise to the liver. Stem-like cells interacted with cancer-associated fibroblasts and endothelial cells via the DLL4-NOTCH signalling pathway to metastasise from primary CRC to the ovary. In the oCRC microenvironment, myofibroblasts provide cancer cells with glutamine and perform a metabolic reprogramming, which may be essential for cancer cells to localise and develop in the ovary. CONCLUSION: We uncover a mechanism for organ-specific CRC metastasis.

Super-enhancer RNA m6A promotes local chromatin accessibility and oncogene transcription in pancreatic ductal adenocarcinoma.

The biological functions of noncoding RNA N6-methyladenosine (m6A) modification remain poorly understood. In the present study, we depict the landscape of super-enhancer RNA (seRNA) m6A modification in pancreatic ductal adenocarcinoma (PDAC) and reveal a regulatory axis of m6A seRNA, H3K4me3 modification, chromatin accessibility and oncogene transcription. We demonstrate the cofilin family protein CFL1, overexpressed in PDAC, as a METTL3 cofactor that helps seRNA m6A methylation formation. The increased seRNA m6As are recognized by the reader YTHDC2, which recruits H3K4 methyltransferase MLL1 to promote H3K4me3 modification cotranscriptionally. Super-enhancers with a high level of H3K4me3 augment chromatin accessibility and facilitate oncogene transcription. Collectively, these results shed light on a CFL1-METTL3-seRNA m6A-YTHDC2/MLL1 axis that plays a role in the epigenetic regulation of local chromatin state and gene expression, which strengthens our knowledge about the functions of super-enhancers and their transcripts.

CSTF2 mediated mRNA N6-methyladenosine modification drives pancreatic ductal adenocarcinoma m6A subtypes.

N6-methyladenosine (m6A) modification of gene transcripts plays critical roles in cancer. Here we report transcriptomic m6A profiling in 98 tissue samples from 65 individuals with pancreatic ductal adenocarcinoma (PDAC). We identify 17,996 m6A peaks with 195 hyper-methylated and 93 hypo-methylated in PDAC compared with adjacent normal tissues. The differential m6A modifications distinguish two PDAC subtypes with different prognosis outcomes. The formation of the two subtypes is driven by a newly identified m6A regulator CSTF2 that co-transcriptionally regulates m6A installation through slowing the RNA Pol II elongation rate during gene transcription. We find that most of the CSTF2-regulated m6As have positive effects on the RNA level of host genes, and CSTF2-regulated m6As are mainly recognized by IGF2BP2, an m6A reader that stabilizes mRNAs. These results provide a promising PDAC subtyping strategy and potential therapeutic targets for precision medicine of PDAC.

LncRNA BCAN-AS1 stabilizes c-Myc via N6-methyladenosine-mediated binding with SNIP1 to promote pancreatic cancer.

C-Myc overexpression contributes to multiple hallmarks of human cancer but directly targeting c-Myc is challenging. Identification of key factors involved in c-Myc dysregulation is of great significance to develop potential indirect targets for c-Myc. Herein, a collection of long non-coding RNAs (lncRNAs) interacted with c-Myc is detected in pancreatic ductal adenocarcinoma (PDAC) cells. Among them, lncRNA BCAN-AS1 is identified as the one with highest c-Myc binding enrichment. BCAN-AS1 was abnormally elevated in PDAC tumors and high BCAN-AS1 level was significantly associated with poor prognosis. Mechanistically, Smad nuclear-interacting protein 1 (SNIP1) was characterized as a new N6-methyladenosine (m6A) mediator binding to BCAN-AS1 via recognizing its m6A modification. m6A-modified BCAN-AS1 acts as a scaffold to facilitate the formation of a ternary complex together with c-Myc and SNIP1, thereby blocking S phase kinase-associated protein 2 (SKP2)-mediated c-Myc ubiquitination and degradation. Biologically, BCAN-AS1 promotes malignant phenotypes of PDAC in vitro and in vivo. Treatment of metastasis xenograft and patient-derived xenograft mouse models with in vivo-optimized antisense oligonucleotide of BCAN-AS1 effectively represses tumor growth and metastasis. These findings shed light on the pro-tumorigenic role of BCAN-AS1 and provide an innovant insight into c-Myc-interacted lncRNA in PDAC.

N 6-methyladenosine Modification of FZR1 mRNA Promotes Gemcitabine Resistance in Pancreatic Cancer.

The therapeutic options for treating pancreatic ductal adenocarcinoma (PDAC) are limited, and resistance to gemcitabine, a cornerstone of PDAC chemotherapy regimens, remains a major challenge. N6-methyladenosine (m6A) is a prevalent modification in mRNA that has been linked to diverse biological processes in human diseases. Herein, by characterizing the global m6A profile in a panel of gemcitabine-sensitive and gemcitabine-insensitive PDAC cells, we identified a key role for elevated m6A modification of the master G0-G1 regulator FZR1 in regulating gemcitabine sensitivity. Targeting FZR1 m6A modification augmented the response to gemcitabine treatment in gemcitabine-resistant PDAC cells both in vitro and in vivo. Mechanistically, GEMIN5 was identified as a novel m6A mediator that specifically bound to m6A-modified FZR1 and recruited the eIF3 translation initiation complex to accelerate FZR1 translation. FZR1 upregulation maintained the G0-G1 quiescent state and suppressed gemcitabine sensitivity in PDAC cells. Clinical analysis further demonstrated that both high levels of FZR1 m6A modification and FZR1 protein corresponded to poor response to gemcitabine. These findings reveal the critical function of m6A modification in regulating gemcitabine sensitivity in PDAC and identify the FZR1-GEMIN5 axis as a potential target to enhance gemcitabine response. SIGNIFICANCE: Increased FZR1 translation induced by m6A modification engenders a gemcitabine-resistant phenotype by inducing a quiescent state and confers a targetable vulnerability to improve treatment response in PDAC.

RNA m6A regulates transcription via DNA demethylation and chromatin accessibility.

Transcriptional regulation, which integrates chromatin accessibility, transcription factors and epigenetic modifications, is crucial for establishing and maintaining cell identity. The interplay between different epigenetic modifications and its contribution to transcriptional regulation remains elusive. Here, we show that METTL3-mediated RNA N6-methyladenosine (m6A) formation leads to DNA demethylation in nearby genomic loci in normal and cancer cells, which is mediated by the interaction between m6A reader FXR1 and DNA 5-methylcytosine dioxygenase TET1. Upon recognizing RNA m6A, FXR1 recruits TET1 to genomic loci to demethylate DNA, leading to reprogrammed chromatin accessibility and gene transcription. Therefore, we have characterized a regulatory mechanism of chromatin accessibility and gene transcription mediated by RNA m6A formation coupled with DNA demethylation, highlighting the importance of the crosstalk between RNA m6A and DNA modification in physiologic and pathogenic process.

ATXN2-mediated translation of TNFR1 promotes esophageal squamous cell carcinoma via m6A-dependent manner.

N6-methyladenosine (m6A) is the most prevalent RNA modification, and the effect of its dysregulation on esophageal squamous cell carcinoma (ESCC) development remains unclear. Here, by performing transcriptome-wide m6A sequencing in 16 ESCC tissue samples, we identified the key roles of m6A in TNFRSF1A (also known as TNFR1)-mediated MAPK and NF-κB activation in ESCC. Mechanistically, a functional protein involved in m6A methylation, ATXN2, is identified that augments the translation of TNFRSF1A by binding to m6A-modified TNFRSF1A mRNA. Upregulation of the TNFRSF1A protein level, a vital upstream switch for TNFRSF1A-mediated signaling events, activates the NF-κB and MAPK pathways and thus promotes ESCC development. Furthermore, TNFRSF1A m6A modifications and protein levels are upregulated in ESCC, and high levels of TNFRSF1A m6A and protein are correlated with poor ESCC patient survival. These results collectively indicate that the m6A-TNFRSF1A axis is critical for ESCC development and thus may serve as a potential druggable target.

Inflammatory cytokine-regulated tRNA-derived fragment tRF-21 suppresses pancreatic ductal adenocarcinoma progression.

The tumorigenic mechanism for pancreatic ductal adenocarcinoma (PDAC) is not clear, although chronic inflammation is implicated. Here, we identified an inflammatory cytokine-regulated transfer RNA-derived (tRNA-derived) fragment, tRF-21-VBY9PYKHD (tRF-21), as a tumor suppressor in PDAC progression. We found that the biogenesis of tRF-21 could be inhibited by leukemia inhibitory factor and IL-6 via the splicing factor SRSF5. Reduced tRF-21 promoted AKT2/1-mediated heterogeneous nuclear ribonucleoprotein L (hnRNP L) phosphorylation, enhancing hnRNP L to interact with dead-box helicase 17 (DDX17) to form an alternative splicing complex. The provoked hnRNP L-DDX17 activity preferentially spliced Caspase 9 and mH2A1 pre-mRNAs to form Caspase 9b and mH2A1.2, promoting PDAC cell malignant phenotypes. The tRF-21 levels were significantly lower in PDACs than in normal tissues, and patients with low tRF-21 levels had a poor prognosis. Treatment of mouse PDAC xenografts or patient-derived xenografts (PDXs) with tRF-21 mimics repressed tumor growth and metastasis. These results demonstrate that tRF-21 has a tumor-suppressive effect and is a potential therapeutic agent for PDAC.

Genome-wide identification and characterization of circular RNA m6A modification in pancreatic cancer.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant modification of RNA in eukaryotic cells and play critical roles in cancer. While most related studies focus on m6A modifications in linear RNA, transcriptome-wide profiling and exploration of m6A modification in circular RNAs in cancer is still lacking. METHODS: For the detection of m6A modification in circRNAs, we developed a new bioinformatics tools called Circm6A and applied it to the m6A-seq data of 77 tissue samples from 58 individuals with pancreatic ductal adenocarcinoma (PDAC). RESULTS: Circm6A performs better than the existing circRNA identification tools, which achieved highest F1 score among these tools in the detection of circRNAs with m6A modifications. By using Circm6A, we identified a total of 8807 m6A-circRNAs from our m6A-seq data. The m6A-circRNAs tend to be hypermethylated in PDAC tumor tissues compared with normal tissues. The hypermethylated m6A-circRNAs were associated with a significant gain of circRNA-mRNA coexpression network, leading to the dysregulation of many important cancer-related pathways. Moreover, we found the cues that hypermethylated m6A-circRNAs may promote the circularization and translation of circRNAs. CONCLUSIONS: These comprehensive findings further bridged the knowledge gaps between m6A modification and circRNAs fields by depicting the m6A-circRNAs genomic landscape of PDAC patients and revealed the emerging roles played by m6A-circRNAs in pancreatic cancer. Circm6A is available at https://github.com/canceromics/circm6a .

N6 -methyladenosine-Mediated Upregulation of WTAPP1 Promotes WTAP Translation and Wnt Signaling to Facilitate Pancreatic Cancer Progression.

Pseudogenes may play important roles in cancer. Here, we explore the mechanism and function of a pseudogene WTAPP1 in the progress of pancreatic ductal adenocarcinoma (PDAC). WTAPP1 RNA was significantly elevated in PDAC and was associated with poor prognosis in patients. Overexpression of WTAPP1 RNA promoted PDAC proliferation and invasiveness in vitro and in vivo. Mechanistically, N 6-methyladenosine (m6A) modification stabilized WTAPP1 RNA via CCHC-type zinc finger nucleic-acid binding protein (CNBP), resulting in increased levels of WTAPP1 RNA in PDAC cells. Excessive WTAPP1 RNA bound its protein-coding counterpart WT1-associated protein (WTAP) mRNA and recruited more EIF3 translation initiation complex to promote WTAP translation. Increased WTAP protein enhanced the activation of Wnt signaling and provoked the malignant phenotypes of PDAC. Decreasing WTAPP1 RNA significantly suppressed the in vivo growth and metastasis of PDAC cell lines and patient-derived xenografts. These results indicate that m6A-mediated increases in WTAPP1 expression promote PDAC progression and thus may serve as a therapeutic target. SIGNIFICANCE: This study reveals how aberrant m6A modification of the WTAPP1 pseudogene results in increased translation of its protein-coding counterpart to promote Wnt signaling, which contributes to pancreatic cancer progression.

NSUN2-mediated RNA 5-methylcytosine promotes esophageal squamous cell carcinoma progression via LIN28B-dependent GRB2 mRNA stabilization.

5-Methylcytosine (m5C) is a posttranscriptional RNA modification participating in many critical bioprocesses, but its functions in human cancer remain unclear. Here, by detecting the transcriptome-wide m5C profiling in esophageal squamous cell carcinoma (ESCC), we showed increased m5C methylation in ESCC tumors due to the overexpressed m5C methyltransferase NSUN2. Aberrant expression of NSUN2 was positively regulated by E2F Transcription Factor 1 (E2F1). High NSUN2 levels predicted poor survival of ESCC patients. Moreover, silencing NSUN2 suppressed ESCC tumorigenesis and progression in Nsun2 knockout mouse models. Mechanistically, NSUN2 induced m5C modification of growth factor receptor-bound protein 2 (GRB2) and stabilized its mRNA, which was mediated by a novel m5C mediator, protein lin-28 homolog B (LIN28B). Elevated GRB2 levels increased the activation of PI3K/AKT and ERK/MAPK signalling. These results demonstrate that NSUN2 enhances the initiation and progression of ESCC via m5C-LIN28B dependent stabilization of GRB2 transcript, providing a promising epitranscriptomic-targeted therapeutic strategy for ESCC.

LINC00842 inactivates transcription co-regulator PGC-1α to promote pancreatic cancer malignancy through metabolic remodelling.

The molecular mechanism underlying pancreatic ductal adenocarcinoma (PDAC) malignancy remains unclear. Here, we characterize a long intergenic non-coding RNA LINC00842 that plays a role in PDAC progression. LINC00842 expression is upregulated in PDAC and induced by high concentration of glucose via transcription factor YY1. LINC00842 binds to and prevents acetylated PGC-1α from deacetylation by deacetylase SIRT1 to form PGC-1α, an important transcription co-factor in regulating cellular metabolism. LINC00842 overexpression causes metabolic switch from mitochondrial oxidative catabolic process to fatty acid synthesis, enhancing the malignant phenotypes of PDAC cells. High LINC00842 levels are correlated with elevated acetylated- PGC-1α levels in PDAC and poor patient survival. Decreasing LINC00842 level and inhibiting fatty acid synthase activity significantly repress PDAC growth and invasiveness in mouse pancreatic xenograft or patient-derived xenograft models. These results demonstrate that LINC00842 plays a role in promoting PDAC malignancy and thus might serve as a druggable target.

Serum piRNA-54265 is a New Biomarker for early detection and clinical surveillance of Human Colorectal Cancer.

Background: Our previous study has demonstrated an oncogenic role of PIWI-interacting RNA-54265 (piR-54265) in colorectal cancer (CRC). Here, we investigate whether it can be a blood biomarker for population screening and clinical applications. Methods: Serum piR-54265 levels were determined by a digital PCR method in 209 cancer-free healthy controls, 725 patients with CRC, 1303 patients with other types of digestive cancer and 192 patients with benign colorectal tumors. A prospective case-control analysis was conducted to assess the predictive value of serum piR-54265 for future CRC diagnosis. Receiver operating characteristic (ROC) curve was constructed to quantify the diagnostic performance of serum piR-54265 levels by assessing its sensitivity, specificity and respective areas under curve (AUC). The odds ratios (ORs) were computed using multivariate logistic regression models. Results: Serum piR-54265 levels were significantly elevated only in patients with CRC compared with controls and patients with other cancer types. The AUC for recognizing CRC was 0.896 (95% CI, 0.874-0.914), with a sensitivity and specificity being 85.7% and 65.1% at 1500 copies/µL as a cut-off value. The serum piR-54265 levels in patients declined substantially after surgery but increased significantly again when tumor relapses. The prediagnostic serum piR-54265 levels were significantly associated with future CRC diagnosis, with the ORs of 7.23, 2.80, 2.45, and 1.24 for those whose CRC was diagnosed within 1, 2, 3 and >3 years. Serum piR-54265 test is more sensitive than other blood CRC markers. Conclusion: Serum piR-54265 may serve as a valuable biomarker for CRC screening, early detection and clinical surveillance.

Excessive miR-25-3p maturation via N6-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression.

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.

PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interaction with SEPW1 pseudogene SEPW1P RNA.

BACKGROUND: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression. METHODS: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels. RESULTS: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer. CONCLUSIONS: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.

PIWI-interacting RNA-54265 is oncogenic and a potential therapeutic target in colorectal adenocarcinoma.

Although PIWI-interacting RNAs (piRNAs) have recently been linked to human diseases, their roles and functions in malignancies remain unclear. This study aimed to investigate the significance of some piRNAs in colorectal cancer (CRC). Methods: We first analyzed the expression profile of piRNAs in CRC using the TCGA and GEO databases. The top 20 highly expressed piRNAs were selected and tested in our CRC tumor and non-tumor tissue samples. We then examined the relevance of the significantly differentially expressed piRNA to the CRC outcomes in 218 patients receiving postoperative chemotherapy and 317 patients receiving neoadjuvant chemotherapy. A series of biochemical and molecular biological assays were conducted to elucidate the functional mechanism of a piRNA of interest in CRC. Furthermore, experiments with mice xenografts were performed to evaluate the therapeutic effect of an inhibitor specific to the piRNA. Results: We found that among the examined 20 piRNAs, only piRNA-54265 was overexpressed in CRC compared with non-tumor tissues and higher levels in tumor or in serum were significantly associated with poor survival in patients. Functional assays demonstrated that piRNA-54265 binds PIWIL2 protein and this is necessary for the formation of PIWIL2/STAT3/phosphorylated-SRC (p-SRC) complex, which activates STAT3 signaling and promotes proliferation, metastasis and chemoresistance of CRC cells. Treatment with a piRNA-54265 inhibitor significantly suppressed the growth and metastasis of implanted tumors in mice. Conclusion: These results indicate that piRNA-54265 is an oncogenic RNA in CRC and thus might be a therapeutic target.