Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of symmetrical dimethylation of arginine residues in proteins. WD repeat domain 77 (WDR77), also known as p44, MEP50, or WD45, ...forms a stoichiometric complex with PRMT5. The PRMT5/p44 complex is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is re-activated during prostate and lung tumorigenesis. The molecular mechanisms by which PRMT5 and p44 promote cellular proliferation are unknown.
Expression of PRMT5 and p44 in lung and prostate cancer cells was silenced and their target genes were identified. The regulation of target genes was validated in various cancer cells during lung development and tumorigenesis. Altered expression of target genes was achieved by ectopic cDNA expression and shRNA-mediated silencing.
PRMT5 and p44 regulate expression of a specific set of genes encoding growth and anti-growth factors, including receptor tyrosine kinases and antiproliferative proteins. Genes whose expression was suppressed by PRMT5 and p44 encoded anti-growth factors and inhibited cell growth when ectopically expressed. In contrast, genes whose expression was enhanced by PRMT5 and p44 encoded growth factors and increased cell growth when expressed. Altered expression of target genes is associated with re-activation of PRMT5 and p44 during lung tumorigenesis.
Our data provide the molecular basis by which PRMT5 and p44 regulate cell growth and lay a foundation for further investigation of their role in lung tumor initiation.
The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and ...hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.
► Identification of miRNomes in human normal liver, hepatitis liver and HCC ► miR-199a/b-3p is the most consistently decreased miRNA in HCC ► Low miR-199-3p expression correlates with poor survival of HCC patients ► miR-199-3p inhibits PAK4/Raf/MEK/ERK prosurvival pathway in HCC
Lenvatinib is an inhibitor of multiple receptor tyrosine kinases that was recently authorized for first-line treatment of hepatocellular carcinoma (HCC). However, the clinical benefits derived from ...lenvatinib are limited, highlighting the urgent need to understand mechanisms of resistance. We report here that HCC cells develop resistance to lenvatinib by activating EGFR and stimulating the EGFR-STAT3-ABCB1 axis. Lenvatinib resistance was accompanied by aberrant cholesterol metabolism and lipid raft activation. ABCB1 was activated by EGFR in a lipid raft-dependent manner, which significantly enhanced the exocytosis of lenvatinib to mediate resistance. Furthermore, clinical specimens of HCC showed a correlation between the activation of the EGFR-STAT3-ABCB1 pathway and lenvatinib response. Erlotinib, an EGFR inhibitor that has also been shown to inhibit ABCB1, suppressed lenvatinib exocytosis, and combined treatment with lenvatinib and erlotinib demonstrated a significant synergistic effect on HCC both in vitro and in vivo. Taken together, these findings characterize a mechanism of resistance to a first-line treatment for HCC and offer a practical means to circumvent resistance and treat the disease.
HCC cells acquire resistance to lenvatinib by activating the EGFR-STAT3-ABCB1 pathway, identifying combined treatment with erlotinib as a strategy to overcome acquired resistance and improve the clinical benefit of lenvatinib.
PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is ...involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.
WDR77 (WD repeat domain 77) is expressed during earlier lung development when cells are rapidly proliferating, but is absent from adult lung. It is re-activated during lung tumorigenesis and is ...essential for lung cancer cell proliferation. Signalling pathways/molecules that control WDR77 gene expression are unknown. Promoter mapping, gel shift assay and ChIP revealed that the WDR77 promoter contains bona fide response elements for E2F and GATA transcriptional factors as demonstrated in prostate cancer, lung cancer and erythroid cells, as well as in mouse lung tissues. The WDR77 promoter is transactivated by E2F1, E2F3, GATA1 and GATA6, but suppressed by E2F6, GATA1 and GATA3 in prostate cancer PC3 cells. WDR77 expression is associated with E2F1, E2F3, GATA2 and GATA6 occupancy on the WDR77 gene, whereas, in contrast, E2F6, GATA1 and GATA3 occupancy is associated with the loss of WDR77 expression during erythroid maturation and lung development. More importantly, the loss of WDR77 expression that results from E2F and GATA switches is required for cellular differentiation of erythroid and lung epithelial cells. In contrast, lung cancer cells avoid post-mitotic differentiation by sustaining WDR77 expression. Altogether, the present study provides a novel molecular mechanism by which WDR77 is regulated during erythroid and lung development and lung tumorigenesis.
•iSEC is applied to determine the intra-fiber pore size distributions of C-CP fibers.•The average pore size of ∼4nm practically excludes peptides/proteins of >∼10,000Da.•van Deemter and Knox plots ...reveal a virtual absence of mass transfer limitations.•Characteristic particle diameters (dp) from Knox equation fitting are ∼ 50μm.
Capillary-channeled polymer (C-CP) fibers have been utilized as liquid chromatography stationary phases, primarily for biomacromolecule separations on the analytical and preparative scales. The collinear packing of the eight-channeled C-CP fibers provides for very efficient flow, allowing operation at high linear velocity (u>100mm s−1) and low backpressure (<2000psi) in analytical-scale separations. To take advantage of these fluid transport properties, there must not be mass transfer limitations as would be imposed by having an appreciably porous phase, wherein solute diffusion limits the overall mass transport rates. To better understand the physical nano-/micro- structure of C-CP fibers, inverse size exclusion chromatography (iSEC) has been employed to determine the pore size distribution (PSD) within C-CP fibers. A diversity of test species (from metal ions to large proteins) was used as probes under non-retaining conditions to obtain a response curve reflecting the apparent partition coefficient (Kd) versus hydrodynamic radii (rm). A mean pore radius (rp) of 4.2nm with standard deviation (sp) of ±1.1nm was calculated by fitting the Kd versus rm data to model equations with a Gaussian pore size distribution, and a pore radius of 4.0±0.1nm was calculated based on a log-normal distribution. The derived mean pore radius is much smaller than traditional support materials, with the standard deviation showing a relatively uniform pore distribution. van Deemter plots were analyzed to provide practical confirmation of the structural implications. Large molecules (e.g., proteins) that are fully excluded from pores have no significant C-terms in the van Deemter plots whereas small molecules that can access the pore volumes display appreciable C-terms, as expected. Fitting of retention data to the Knox equation suggests that the columns operate with a characteristic particle diameter (dp) of ∼53μm.
Protein arginine methyltransferase 5 (PRMT5) plays multiple roles in a large number of cellular processes, and its subcellular localization is dynamically regulated during mouse development and ...cellular differentiation. However, little is known of the functional differences between PRMT5 in the cytoplasm and PRMT5 in the nucleus. Here, we demonstrated that PRMT5 predominantly localized in the cytoplasm of prostate cancer cells. Subcellular localization assays designed to span the entire open-reading frame of the PRMT5 protein revealed the presence of three nuclear exclusion signals (NESs) in the PRMT5 protein. PRMT5 and p44/MED50/WD45/WDR77 co-localize in the cytoplasm, and both are required for the growth of prostate cancer cells in an PRMT5 methyltransferase activity-dependent manner. In contrast, PRMT5 in the nucleus inhibited cell growth in a methyltransferase activity-independent manner. Consistent with these observations, PRMT5 localized in the nucleus in benign prostate epithelium, whereas it localized in the cytoplasm in prostate premalignant and cancer tissues. We further found that PRMT5 alone methylated both histone H4 and SmD3 proteins but PRMT5 complexed with p44 and pICln methylated SmD3 but not histone H4. These results imply a novel mechanism by which PRMT5 controls cell growth and contributes to prostate tumorigenesis.
Long noncoding RNAs (lncRNAs) are versatile in functions and can regulate cancer development, including the modulation of cancer immunity. Immune-related lncRNA signatures predicting prognosis have ...been reported in multiple cancers, but relevant studies in gastric cancer (GC) are still lacking.
We performed a comprehensive analysis using TCGA and Immport databases and identified an immune-related lncRNA signature by univariate and multivariate Cox regression analysis. qRT-PCR and immunohistochemistry assays were used for further validation. KEGG and GO analysis and ceRNA network establishment were carried out to explore the regulatory functions.
We first identified an immune-related lncRNA signature, which can stratify gastric cancer patients into high- and low-risk subgroups and the high-risk cases frequently suffered from shorter overall survival time. Next, we validated the reliability of the lncRNA signature in an independent 75 gastric cancer samples and demonstrated that the three-year survival rate in high-risk patients was only 30.8%
66.5% in low-risk counterparts. Functional exploration indicated that the lncRNA signature might participate in multiple cancer-associated processes including cell adhesion and migration, cytokine-receptor interaction and immune evasion. Additionally, we observed that high-risk samples tended to form an immunosuppressive microenvironment, which had more M2-polarized macrophages and Tregs, but fewer CD8 effector T cells within tumors. Moreover, we found that PD-1 and PD-L1 were dramatically upregulated in a subset of high-risk patients with abundant M2 and Treg infiltration, implying these patients may benefit from anti-PD-1 and PD-L1 immunotherapy.
These results showed that the immune-related lncRNA signature had a prominent capacity to predict overall survival and the immune status of microenvironment in gastric cancer. Our findings may be useful for the risk-stratification management and provide a valuable clue to identify proper patients potentially benefit from immune checkpoint therapy in gastric cancer.
Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver cancers and is one of the leading causes of cancer-related death in many countries. Cancer cell-derived exosomes are shown ...to mediate communications between cancer cells and the microenvironment, promoting tumorigenesis. Hedgehog signaling pathway plays important roles in cancer development of HCC.
Exosomes were isolated from culture medium of HCC cell lines PLC/PRF/5 and MHCC-97H and were found to promote cancer cell growth measured with cell proliferation and colony formation assay. HCC cells cultured with cancer cell-derived exosome had increased cancer stem cell (CSC) population demonstrated by increased cell sphere formation CSC marker expressions. Hedgehog protein Shh was found to be highly expressed in these two HCC cell lines and preferably carried by exosomes. When Shh was knocked down with shRNA, the resulting exosomes had a reduced effect on promoting cancer cell growth or CSC population increase compared to normal cell-derived exosomes.
The ability of PLC/PRF/5 cells to form tumor in a xenograft model was increased by the addition of the exosomes from control cancer cells but not the exosomes from Shh knocked down cancer cells. Finally, the higher plasma Exo-Shh levels were associated with later tumor stages, higher histological grades, multiple tumors, and higher recurrence rates.
This study demonstrated that HCC cells secreted Shh
exosome and promote tumorigenesis through the activated Hedgehog pathway.
Currently, chemokines and their receptors, CXCL12-CXCR4 and CCL21-CCR7 axes, are deemed vital factors in the modulation of angiogenesis and are crucial for the growth and development of liver cancer. ...Tumor-derived DNA can be recognized by immune cells to induce an autoimmune response. In this study, we demonstrated the mechanism of tumor-derived DNA on the CXCL12-CXCR4 and CCL21-CCR7 axes of hepatocellular carcinoma (HCC) cells and the regulatory effect of sinomenine hydrochloride. Tumor-derived DNA was separated from HCCLM cell lines. Tumor-derived DNA was transfected into SK-Hep1 cells by Lipofectamine 2000. We found that sinomenine hydrochloride reduced the expression of CXCR4, CXCR12, CCR7, and CCL21 in HCC cells, suppressed the growth and invasion of HCC cells, and increased apoptosis. In contrast to the controls, the protein expressions of CXCR4, CXCL12, CCR7, CCL21, P-ERK1/2, MMP-9, and MMP-2 in SK-Hep1 cells were significantly increased after transfection of tumor-derived DNA, while the increase was reversed by sinobine hydrochloride. Acid sinomenine interferes with tumor-derived DNA and affects ERK/MMP signaling
via
the CXCL12/CXCR4 axis in HCC cells. CXCR4 siRNA and CCR7 siRNA attenuated tumor-derived DNA activation of ERK1/2/MMP2/9 signaling pathways in HCC cells. CXCR4-oe and CCR7-OE enhance the stimulation of erK1/2/MMP2/9 signaling pathway by tumor-derived DNA in HCC cells. Tumor-derived DNA reduced apoptosis and increased invasion of SK-Hep1 cells by CXCL12-CXCR4 axis and CCL21-CCR7 axis, and sinobine hydrochloride reversed this regulation. These results strongly suggest that tumor-derived DNA can increase the growth and invasion of oncocytes
via
the upregulation of the expression of CXCL12-CXCR4 and CCL21-CCR7 axis and through ERK1/2/MMP2/9 signaling pathway in HCC cells, and sinobine hydrochloride can inhibit this signaling pathway, thus inhibiting HCC cells. These results provide new potential therapeutic targets for blocking the progression of HCC induced by CXCL12-CXCR4 axis and CCL21-CCR7.
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