Commonly observed in colorectal cancer is the elevated expression of the prostaglandin (PG) synthase COX-2. In normal intestinal epithelium, the COX-2 mRNA is targeted for rapid decay through the ...3'-untranslated region (3'-UTR) adenylate- and uridylate (AU)-rich element (ARE), whereas in tumors ARE-mediated decay is compromised. Here we show that the COX-2 ARE can mediate degradation through microRNA (miRNA)-mediated regulation. We identified miR-16 to bind the COX-2 3'-UTR and inhibit COX-2 expression by promoting rapid mRNA decay. In colorectal cancer cells and tumors, miR-16 levels were decreased approximately twofold and miR-16 expression in cancer cells attenuated COX-2 expression and PG synthesis. The COX-2 ARE is also bound by the RNA-binding protein HuR. In colorectal cancer tumors, HuR is overexpressed and localized within the cytoplasm, where it promotes ARE-mRNA stabilization. Under conditions of HuR overexpression, miR-16 was unable to promote rapid mRNA decay through the COX-2 ARE. Ribonucleoprotein immunoprecipitation of HuR showed direct association with miR-16 that was reversed when cytoplasmic trafficking of HuR was inhibited. Furthermore, this interaction between HuR and miR-16 promoted the downregulation of miR-16. These new results identify miR-16 as a central posttranscriptional regulator of COX-2 and show the ability of elevated levels of HuR to antagonize miR-16 function. Along with insight into altered ARE-mediated mRNA decay observed in colorectal cancer, these findings provide a new explanation for tumor-derived loss of miR-16.
Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected ...siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.
This detailed characterization of membrane association in siRNA‐mediated gene silencing establishes the rough ER as major site for canonical RISC loading and target RNA cleavage.
Tumor buds in colorectal cancer are hypothesized to undergo a (partial) epithelial-mesenchymal transition (EMT). If so, cytokeratin (CK) and vimentin (VIM) co-expression is expected. CK+/VIM+ can ...also be found in some stromal cells; however, their origin remains unclear. Here, we determine the frequency of CK+/VIM+ tumor cells and characterize the CK+/VIM+ stroma in colorectal cancer. Three cell populations (CK+, VIM+, CK+/VIM+) were sorted using DepArray and fluorescence-activated cell sorting (FACS). Tumor areas were selected to include tumor center, stroma and tumor budding. Fluorescence microscopy was used to visualize co-expressing cells on whole slides. A next-generation tissue microarray (ngTMA) of matched Pan-CK–positive and -negative stroma was constructed and stained for E-cadherin, VIM, Snail1, Twist1, Zeb1 and Zeb2, COL11A1, SPARC, CD90, α-SMA, FAP and WT1. CK+/VIM+ co-expressing tumor cells were detected using all three methods. With DepArray, only tumor budding areas contained CK+/VIM+ cells. The proportion of CK+/VIM+ tumor cells was low (1.5%–22%). CK+ stroma was associated with aggressive tumor features like distant metastasis (P = .0003), lymphatic invasion (P = .0009) and tumor budding (P = .0084). CK+/VIM+ stroma was characterized by positive WT1 (P < .001), ZEB2 (P < .001), TWIST1 (P = .009), and FAP (P = .003). Our data suggest that CK+/VIM+ tumor cells exist, albeit in low numbers and could represent a subgroup of tumor buds in partial EMT. CK+/VIM+ stroma may be of mesothelial origin and shows features of mesenchymal cells and cancer-associated fibroblasts. These results, together with the association with metastasis point to cells in mesothelial-mesenchymal transition (MMT). This atypical stroma may be a potential target for therapy.
•CK+/VIM+ tumor cells exist in colorectal cancer, as shown using DepArray, FACS, and confocal microscopy and are located in tumor budding regions.•These CK+/VIM+ tumor cells comprise 1.5% to 22% of the CK-positive cell population.•CK+/VIM+ stroma is associated with aggressive tumor behavior including metastasis.•This double-positive stroma expresses significantly more WT1, ZEB2, TWIST1 and FAP compared to CK-negative tumor-associated stroma but shows no difference in E-cadherin, SNAIL1, ZEB1, COL11A1, SPARC, CD90, or α-SMA.•These results suggest that double-positive fibroblasts are experiencing mesothelial-mesenchymal transition (MMT).
Tumor-associated macrophages (TAM) play a controversial role in epithelial-mesenchymal transition (EMT) and prognosis of colorectal cancer (CRC). In particular, the microlocalization, polarization ...and prognostic impact of TAM in the immediate environment of invading CRC cells has not yet been established. To address this clinically relevant question, intraepithelial (iCD68) and stromal macrophages (sCD68), M1-macrophages (iNOS), M2-macrophages (CD163), cytokeratin-positive cancer cells (tumor buds) and expression of the anti-phagocytic marker CD47 were investigated in primary tumors of 205 well-characterized CRC patients. Cell-to-cell contacts between tumor buds and TAM were detected using high-resolution digital scans. The composition of the tumor microenvironment was analyzed with clinicopathological and molecular features. High CD68 counts predicted long term overall survival independent of microlocalization (iCD68 p=0.0016; sCD68 p=0.03), pT, pN, pM and post-operative therapy. CD68 infiltration correlated with significantly less tumor budding (iCD68 p=0.0066; sCD68 p=0.0091) and absence of lymph node metastasis (sCD68 p=0.0286). Cell-to-cell contact of sCD68 and invading cancer cells was frequent and ameliorated the detrimental prognostic effect of the tumor budding phenotype. Subgroup analysis identified long-term survival with CD47 loss and predominance of CD163
+
M2 macrophages (p = 0.0366). CD163
+
macrophages represented 40% of the total population, and positively correlated with total CD68 macrophage numbers (rCD68/CD163 = 0.32; p = 0.0001). Strong CD163 infiltration predicted lower tumor grade (p = 0.0026) and less lymph node metastasis (p = 0.0056). This study provides direct morphological evidence of an interaction between TAM and infiltrating cancer cells. The prognostic impact of TAM is modulated by phenotype, microlocalization and the expression of anti-phagocytic markers in CRC.
In colorectal cancer (CRC), RHAMM is an independent adverse prognostic factor. The aim of the study was therefore to investigate on the role of RHAMM as a potential direct driver of cell ...proliferation and migration in CRC cell lines and to identify pathways dependent on RHAMM in human CRC. Proliferation, cell cycle alterations and invasive capacity were tested in two RHAMM- and control- knockdown CRC cell lines by flow cytometry and
assays. Tumorigenicity and metastasis formation was assessed in immunodeficient mice. RNA-Seq and immunohistochemistry was performed on six RHAMM+/- primary CRC tumors.
, silencing of RHAMM inhibited CRC cell migration and invasion by 50% (p<0.01).
, RHAMM knockdown resulted in slower growth, lower tumor size (p<0.001) and inhibition of metastasis (p<0.001). Patients with RHAMM-high CRC had a worse prognosis (p=0.040) and upregulated pathways for cell cycle progression and adhesion turnover. RHAMM overexpression is correlated with increased migration and invasion of CRC cells, leads to larger, fast growing tumors, and its downregulation essentially abolishes metastasis in mouse models. RHAMM is therefore a promising therapeutic target in all CRC stages as its inhibition affects growth and dissemination of the primary CRC as well as the metastases.
The immune system plays a pivotal role in the development and progression of colorectal cancer (CRC). Tumor immune rejection has been previously linked to the activation of the interferon-stimulated ...genes (ISG) STAT1, IRF-5 and IRF-1. Specific immunoregulatory microRNAs (miRNAs) may impact the expression of these ISG in the tumor microenvironment. In this translational study, we develop a digital image analysis protocol to identify the ISG-gene expression signature and investigate miRNA expression in the immediate environment of invading cancer cells. Digital immunophenotyping was performed using next generation tissue microarrays from 241 well-characterized CRC patients and analyzed with clinicopathological and molecular information. Active ISG signaling in the tumor stroma differentiated an immune-activated (n = 178) and a quiescent (n = 43) phenotype. The activated phenotype was associated with high counts of intratumoral CD8
+
cytotoxic T-lymphocytes (CTL; p = 0.007) and expression of the immune effector molecules granzyme B (p < 0.001) and perforin (p = 0.020). Immune-activated tumors also showed an elevated expression of the intercellular adhesion molecule-1 (ICAM-1, p = 0.006) which may facilitate CTL infiltration. Patients with immune-activated CRC had a considerably reduced risk of developing distant metastases (p = 0.001, OR = 0.034, 95%CI = 0.006-0.183). High expression of the immunoregulatory miR-34a and miR-93 corresponded to a 2-2.5-fold decrease of STAT1 (p = 0.006) and IRF-1 (p = 0.058), a feature more commonly seen in a quiescent microenvironment. Analysis of a combined ISG marker profile by digital pathology stratifies CRC patients into diametrically opposed immune phenotypes. Targeted inhibition of miRNAs within the tumor microenvironment may form a new strategy to stimulate the anti-tumoral immune response.
The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and ...prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12.
Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular ...responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.
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•A framework for investigating fasting-feeding cycles and circadian rhythms•Time-series analysis shows molecular oscillations are set by fasting-feeding cycles•In-depth lipidomics provides insights into metabolic crosstalk between tissues•Diurnal regulation of the hepatic PE methylation pathway and lipoprotein secretion
Sprenger et al. generate a time-resolved multi-omics resource of liver and plasma and find that molecular oscillations are entrained by fasting-feeding cycles. The authors go on to uncover that the hepatic PE methylation pathway is diurnally regulated and gives rise to two pools of oscillating PC molecules in the circulation.
Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test ...whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival.
A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging.
A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85).
Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC.
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