Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion.

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.

CD206 modulates the role of M2 macrophages in the origin of metastatic tumors.

Tumor metastasis is a key factor affecting the life of patients with malignant tumors. For the past hundred years, scientists have focused on how to kill cancer cells and inhibit their metastasis in vivo, but few breakthroughs have been made. Here we hypothesized a novel mode for cancer metastasis. We show that the phagocytosis of apoptotic tumor cells by macrophages leads to their polarization into the M2 phenotype, and that the expression of stem cell related as well as drug resistance related genes was induced. Therefore, it appears that M2 macrophages have "defected" and have been transformed into the initial "metastatic cancer cells", and thus are the source, at least in part, of the distal tissue tumor metastasis. This assumption is supported by the presence of fused cells with characteristics of both macrophage and tumor cell observed in the peripheral blood and ascites of patients with ovarian cancer. By eliminating the expression of CD206 in M2 macrophages using siRNA, we show that the growth and metastasis of tumors was suppressed using both in vitro cell line and with experimental in vivo mouse models. In summary, we show that M2 macrophages in the blood circulation underwent a "change of loyalty" to become "cancer cells" that transformed into distal tissue metastasis, which could be suppressed by the knockdown of CD206 expression.

HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy.

The search for effective combination therapy with immune checkpoint inhibitors (ICI) has become important for cancer patients who do not respond to the ICI well. Histone deacetylases (HDACs) inhibitors have attracted wide attention as anti-tumor agents. ACY-1215 is a selective inhibitor of HDAC6, which can inhibit the growth of a variety of tumor. We previously revealed that HDAC family is highly expressed in colorectal cancer specimens and mouse models. In this study, ACY-1215 was combined with anti-PD1 to treat tumor-bearing mice associated with colorectal cancer. ACY-1215 combined with anti-PD1 effectively inhibited the colorectal tumor growth. The expression of PD-L1 in tumor of mice were inhibited by ACY-1215 and anti-PD1 combination treatment, whereas some biomarkers reflecting T cell activation were upregulated. In a co-culture system of T cells and tumor cells, ACY-1215 helped T cells to kill tumor cells. Mechanically, HDAC6 enhanced the acetylation of STAT1 and inhibited the phosphorylation of STAT1, thus preventing STAT1 from entering the nucleus to activate PD-L1 transcription. This study reveals a novel regulatory mechanism of HDAC6 on non-histone substrates, especially on protein acetylation. HDAC6 inhibitors may be of great significance in tumor immunotherapy and related combination strategies.

The Value of Microbes in Cancer Neoantigen Immunotherapy.

Tumor neoantigens are widely used in cancer immunotherapy, and a growing body of research suggests that microbes play an important role in these neoantigen-based immunotherapeutic processes. The human body and its surrounding environment are filled with a large number of microbes that are in long-term interaction with the organism. The microbiota can modulate our immune system, help activate neoantigen-reactive T cells, and play a great role in the process of targeting tumor neoantigens for therapy. Recent studies have revealed the interconnection between microbes and neoantigens, which can cross-react with each other through molecular mimicry, providing theoretical guidance for more relevant studies. The current applications of microbes in immunotherapy against tumor neoantigens are mainly focused on cancer vaccine development and immunotherapy with immune checkpoint inhibitors. This article summarizes the related fields and suggests the importance of microbes in immunotherapy against neoantigens.

Epstein-Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase â ¢ endonuclease DICER1.

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis in vivo and in vitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

Establishment of an optimized CTC detection model consisting of EpCAM, MUC1 and WT1 in epithelial ovarian cancer and its correlation with clinical characteristics.

Objective: Emerging studies have demonstrated the promising clinical value of circulating tumor cells (CTCs) for diagnosis, disease assessment, treatment monitoring and prognosis in epithelial ovarian cancer. However, the clinical application of CTC remains restricted due to diverse detection techniques with variable sensitivity and specificity and a lack of common standards. Methods: We enrolled 160 patients with epithelial ovarian cancer as the experimental group, and 90 patients including 50 patients with benign ovarian tumor and 40 healthy females as the control group. We enriched CTCs with immunomagnetic beads targeting two epithelial cell surface antigens (EpCAM and MUC1), and used multiple reverse transcription-polymerase chain reaction (RT-PCR) detecting three markers (EpCAM, MUC1 and WT1) for quantification. And then we used a binary logistic regression analysis and focused on EpCAM, MUC1 and WT1 to establish an optimized CTC detection model. Results: The sensitivity and specificity of the optimized model is 79.4% and 92.2%, respectively. The specificity of the CTC detection model is significantly higher than CA125 (92.2% vs. 82.2%, P=0.044), and the detection rate of CTCs was higher than the positive rate of CA125 (74.5% vs. 58.2%, P=0.069) in early-stage patients (stage I and II). The detection rate of CTCs was significantly higher in patients with ascitic volume ≥500 mL, suboptimal cytoreductive surgery and elevated serum CA125 level after 2 courses of chemotherapy (P<0.05). The detection rate of CTCEpCAM + and CTCMUC1+ was significantly higher in chemo-resistant patients (26.3% vs. 11.9%; 26.4% vs. 13.4%, P<0.05). The median progression-free survival time for CTCMUC1+ patients trended to be longer than CTCMUC1- patients, and overall survival was shorter in CTCMUC1+ patients (P=0.043). Conclusions: Our study presents an optimized detection model for CTCs, which consists of the expression levels of three markers (EpCAM, MUC1 and WT1). In comparison with CA125, our model has high specificity and demonstrates better diagnostic values, especially for early-stage ovarian cancer. Detection of CTCEpCAM+ and CTCMUC1+ had predictive value for chemotherapy resistance, and the detection of CTCMUC1+ suggested poor prognosis.

Overexpression of DUSP6 enhances chemotherapy-resistance of ovarian epithelial cancer by regulating the ERK signaling pathway.

Objective: DUSP6 is a negative regulator of the ERK signaling pathway and plays an important role in chemotherapy-resistance. Previously we showed that DUSP6 is overexpressed in ovarian cancer side population (SP) cells that possess cancer stem cell-like properties and are quiescent and chemotherapy-resistant. Here, we explore the effects of DUSP6 on chemotherapy-resistance by examining its regulation of the ERK signaling pathway and G0/G1 cell cycle arrest. Methods: mRNA and protein expression of DUSP6 and G0/G1 cell cycle checkpoint regulating proteins (CyclinD1, CyclinD3 and CyclinE2) was evaluated among ovarian cancer cell lines and tissue samples. Ovarian cancer cells were transiently transfected to overexpress DUSP6. After treatment with cisplatin, cell viability was measured by the MTS assay at 48 hours and the half maximal inhibitory concentration (IC50) for each cell line was calculated. Subcellular localization and cell cycle analysis were determined by using immunofluorescence and FACS, respectively. Results: SKOV3 and OVCAR8 SP cells were shown to express higher levels of DUSP6 and lower levels of CyclinD3 compared with non-SP (NSP) cells (P<0.001). Among 39 ovarian cancer tissue samples, expression of DUSP6 in the chemotherapy-resistant group (12 samples) was higher than in the chemotherapy-sensitive group (27 samples) (P<0.05). While a lower level of expression of CyclinD3 was seen in the chemotherapy-resistant group, it was not statistically different from the chemotherapy-sensitive group. HO8910 cells where shown to have higher IC50 to cisplatin than SKOV3 or OVCAR8 cells, and this correlated with higher levels of DUSP6 expression. Overexpression of DUSP6 in SKOV3 cells led to an increase in cisplatin IC50 values (P<0.05), and also markedly reduced the expression levels of phospho-ERK1/2 and CyclinD3 and to the predominance of cells in the G0/G1 phase. Conclusion: Our findings reveal an enhancement of chemotherapy-resistance and a predominance of cells in G1 cell cycle arrest in DUSP6-overexpressing ovarian cancer cells. This suggests that overexpression of DUSP6 promotes chemotherapy-resistance through the negative regulation of the ERK signaling pathway, increasing the G0/G1 phase ratio among ovarian cancer cells, and leading to cellular quiescence.

Apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages and promote the proliferation and migration of ovarian cancer cells by activating the ERK signaling pathway.

Ovarian cancer has a high rate of recurrence, with M2 macrophages having been found to be involved in its progression and metastasis. To examine the relationship between macrophages and ovarian cancer in the present study, M0 macrophages were stimulated with apoptotic SKOV3 cells and it was found that these macrophages promoted tumor proliferation and migration. Subsequently, the mRNAs and proteins expressed at high levels in these M2 macrophages were examined by RNA­Seq and quantitative proteomics, respectively, which revealed that M0 macrophages stimulated by apoptotic SKOV3 cells also expressed M2 markers, including CD206, interleukin­10, C­C motif chemokine ligand 22, aminopeptidase­N, disabled homolog 2, matrix metalloproteinase 1 and 5'­nucleotidase. The abundance of phosphorylated Erk1/2 in these macrophages was increased. The results indicate that apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages by activating the ERK pathway. These results suggest possible treatments for patients with ovarian cancer who undergo chemotherapy; inhibiting M2 macrophage differentiation during chemotherapy may reduce the rate of tumor recurrence.