Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored.Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines.The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ-resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling.Together, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC.
Liver metastasis occurs frequently in patients with pancreatic cancer. We analyzed the molecular profiling in liver metastatic lesions aiming to uncover novel genes responsible for tumor progression. Bioinformatics analysis was applied to identify genes directing liver metastasis. CRISPR/Cas9 technology was used to knock out the candidate gene. Proliferation assays, colony formation assays, cell cycle analysis, migration assays, wound healing assays, Immunofluorescence analysis, and the tumor xenograft model of intrasplenic injection were adopted to evaluate the effects of PCSK6 inactivation on cell growth, migration and liver metastasis. GSEA and Western blot were used to investigate the corresponding signaling pathway. PCSK6 was one of the obtained liver-metastasis-related genes in pancreatic cancer. PCSK6 inactivation inhibited cell growth and cell migration, due to G0/G1 cell cycle arrest and the remodeling of cell-cell junctions or the cell skeleton, respectively. PCSK6 inactivation led to fewer counts and lower outgrowth rates of liver metastatic niches in vivo. The Raf-MEK1/2-ERK1/2 axis was repressed by PCSK6 inactivation. Accordingly, we found PCSK6 inactivation could inhibit cell growth, cell migration, and liver metastasis, and explored the role of the Raf-MEK1/2-ERK1/2 axis in PCSK6 inactivation. PCSK6-targeted therapy might represent a novel approach for combatting liver metastasis in pancreatic cancer.
The limitations of optical microscopy to determine the cellular localization of label-free nanoparticles prevent a solid prediction of the cellular effect of particles intended for medical applications. To avoid the strong physicochemical changes associated with fluorescent labelling, which often result in differences in cellular uptake, efficiency and toxicity of particles, novel detection techniques are required.In the present study, we determined the intracellular content of unlabeled SPIONs by analyzing refractive index (RI)-based images from holotomographic three-dimensional (3D) microscopy and side scatter data measured by flow cytometry. The results were compared with the actual cellular SPION amount as quantified by atomic emission spectroscopy (AES).Live cell imaging by 3D holotomographic microscopy demonstrated cell-specific differences in intracellular nanoparticle uptake in different pancreatic cell lines. Thus, treatment of PANC-1SMAD4 (1-4) and PANC-1SMAD4 (2-6) with SPIONs resulted in a significant increase in number of areas with higher RI, whereas in PANC-1, SUIT-2 and PaCa DD183, only a minimal increase of spots with high RI was observed. The increase in areas with high RI was in accordance with the SPION content determined by quantitative iron measurements using AES. In contrast, determination of the SPION amount by flow cytometry was strongly cell type-dependent and did not allow the discrimination between intracellular and membrane-bound SPIONs. However, flow cytometry is a very rapid and reliable method to assess the cellular toxicity and allows an estimation of the cell-associated SPION content.Holotomographic 3D microscopy is a useful method to distinguish between intracellular and membrane-associated particles. Thus, it provides a valuable tool for scientists to evaluate the cellular localization and the particle load, which facilitates prediction of potential toxicity and efficiency of nanoparticles for medical applications.
Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.
Abstract Since their discovery, microRNAs (miRNAs) have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity for in vitro and in vivo applications could lead to a better understanding of their biological role in physiology and disease.
Abstract Epigenetics including DNA and RNA modifications have always been the hotspot field of life sciences in the post-genome era. Since the first mapping of N6-methyladenosine (m 6 A) and the discovery of its widespread presence in mRNA, there are at least 160-170 RNA modifications have been discovered. These methylations occur in different RNA types, and their distribution is species-specific. 5-methylcytosine (m 5 C) has been found in mRNA, rRNA and tRNA of representative organisms from all kinds of species. As reversible epigenetic modifications, m 5 C modifications of RNA affect the fate of the modified RNA molecules and play important roles in various biological processes including RNA stability control, protein synthesis, and transcriptional regulation. Furthermore, accumulative evidence also implicates the role of RNA m 5 C in tumorigenesis. Here, we review the latest progresses in the biological roles of m 5 C modifications and how it is regulated by corresponding “writers”, “readers” and “erasers” proteins, as well as the potential molecular mechanism in tumorigenesis and cancer immunotherapy.
Pancreatic cancer (PaCa) is the fourth leading cause of cancer-related death, and personalized targeted cancer therapy is becoming a promising treatment strategy for PaCa. The central approach of targeted therapy is to find a targetable key and an effective targeting method. In this study, the importance of GPRC5a (the G-protein-coupled receptor family C, member 5, group A) was identified using data mining methods based on published datasets. After analysis of the basic expression of GPRC5a in normal pancreas tissue and various PaCa cell lines, gene editing of GPRC5a in the human PaCa cell line MIA PaCa-2 and the mouse PaCa cell line TB32047 was performed using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins 9) to investigate the influence of GPRC5a on the proliferation and migration of PaCa cells as well as its effects on chemotherapy drug resistance. The results showed that GPRC5a was upregulated in PaCa tissues and various PaCa cell lines. Knockout of GPRC5a reduced the proliferation and migration ability of PaCa cell lines and suppressed the chemotherapy drug resistance of gemcitabine, oxaliplatin, and fluorouracil in PaCa cells. The phosphorylation of GSK-3β (Glycogen synthase kinase-3β) was found to be upregulated in the MIA PaCa-2 and TB32047 cells after GPRC5a knockout. In conclusion, GPRC5a was upregulated in PaCa leading to an enhanced drug resistance in PaCa cells. These results provide for the first time a theoretical basis for the development of an improved PaCa targeted therapy.
Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive digestive malignancy due to frequent late-stage diagnosis, rapid progression and resistance to therapy. With increasing PDAC incidence worldwide, there is an urgent need for new prognostic biomarkers and therapy targets. Recently, RNA methylation has emerged as a new tumorigenic mechanism in different cancers. 5-methylcytosine (m5C) is one of the most frequent RNA modifications and occurs on a variety of RNA species including mRNA, thereby regulating gene expression. Here we investigated the prognostic role of m5C-regulator-associated transcriptional signature in PDAC. We evaluated m5C-regulator status and expression in 239 PDAC samples from publicly available datasets. We used unsupervised consensus clustering analyses to classify PDACs based on m5C-regulator expression. From the resulting signature of differentially expressed genes (DEGs), we selected prognosis-relevant DEGs to stratify patients and build a scoring signature (m5C-score) through LASSO and multivariate Cox regression analyses. The m5C-score represented a highly significant independent prognostic marker. A high m5C-score correlated with poor prognosis in different PDAC cohorts, and was associated with the squamous/basal subtype as well as activated cancer-related pathways including Ras, MAPK and PI3K pathways. Furthermore, the m5C-score correlated with sensitivity to pathway-specific inhibitors of PARP, EGFR, AKT, HER2 and mTOR. Tumors with high m5C-score were characterized by overall immune exclusion, low CD8+ T-cell infiltration, and higher PD-L1 expression. Overall, the m5C-score represented a robust predictor of prognosis and therapy response in PDAC, which was associated with unfavorable molecular subtypes and immune microenvironment.
Pancreatic cancer is one of the most common and fatal cancer. The poor prognosis of pancreatic cancer has not significant improved in the past decades. The main treatments for pancreatic are surgery, radiation therapy and chemotherapy, but there is also a lot of efforts to improve treatments. In recent year, CRISPR/Cas9 technology become one of the most famous and powerful gene editing tools, and has been applied in pancreatic cancer research. It has great potential for scientific research and clinical treatment in pancreatic cancer. In this review, we summarize currently application of CRISPR/Cas9 technology in pancreatic cancer research, and also highlight the potential vulnerable target genes in pancreatic cancer.
ABSTRACT Since their discovery, microRNAs (miRNA)s have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs’ endogenous levels and activity could lead to a better understanding of their biological role in physiology and disease.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)