Yanhong Ji

IL-6, IL-8 and TNF-α levels correlate with disease stage in breast cancer patients

BACKGROUND Breast cancer is the most common cancer in Chinese women. Inflammation contributes to tumor progression and can be induced by excessive production of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). However, how their levels relate to the expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2) by the tumor has not been investigated. OBJECTIVES The aim of the study is to more fully understand the significance of serum IL-6, IL-8 and TNF-α in breast cancers with different ER, PR and HER2 status. MATERIAL AND METHODS Preoperative serum samples were collected from 110 patients diagnosed with ductal carcinoma and 30 healthy control subjects. IL-6, IL-8 and TNF-α levels were determined by enzyme-linked immunosorbent assay (ELISA). Associations of cytokine levels with clinical tumor stage were evaluated, and correlations of serum cytokine levels with ER, PR and HER2 expression were determined using the Pearson correlation coefficient. RESULTS Serum levels of IL-6 and IL-8 were significantly higher in the subjects with ductal carcinoma than in the controls, and strongly correlated with clinical tumor stage, lymph node metastasis, and ER and HER2 antigen expression (p < 0.05). TNF-α levels in stage III carcinoma patients were significantly higher than in the controls (p < 0.01) and were associated with lymph node metastasis (p < 0.01). A strong positive correlation was found between IL-8 and TNF-α levels in the cancer patients (p < 0.0001). CONCLUSIONS The study showed that IL-6, IL-8 and TNF-α levels correlated with clinical disease stage and lymph node metastasis as well as with ER and HER2 antigen expression. Specifically, IL-6 and IL-8 seem to have significant potential as prognostic cancer biomarkers. Analyzing serum cytokine levels might help identify patients with a poor prognosis who may benefit from more aggressive disease management.

TIGIT overexpression diminishes the function of CD4 T cells and ameliorates the severity of rheumatoid arthritis in mouse models

Rheumatoid arthritis (RA) is an immune-mediated disease with a pathogenesis that involves CD4 T cell activation. Multiple immune regulatory molecules expressed on CD4(+) T cells were involved in RA pathogenesis. In this study, we investigated the role of T cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain (TIGIT) in RA. The frequency of TIGIT-positive CD4(+) T cells in the synovial fluid (SF) of active RA patients was lower than that of inactive RA patients. And a negative correlation between RA disease activity and TIGIT expression was found. In CD4(+) T cells isolated from SF of active RA patients, TIGIT upregulation significantly decreased cell proliferation, as shown by MTT assay. TIGIT overexpression also significantly decreased the production of IFN-γ and IL-17, and increased that of IL-10, as determined by ELISA and qRT-PCR. In CD4(+) T cells isolated from SF of inactive RA patients, TIGIT was silenced by siRNA transfection. As expected, TIGIT knockdown resulted in an opposite effect on cell proliferation and the production of cytokines, including IFN-γ, IL-17 and IL-10. A RA mouse model was established using type II collagen induction. TIGIT was upregulated in RA mouse by lentivector infection. As expected, TIGIT overexpression in vivo significantly alleviated the disease severity and deceased the levels of anti-collagen II antibodies. TIGIT upregulation in the early stage was more effective to alleviate disease severity. Our data suggested the potential therapeutic role of TIGIT in RA patients.

Interleukin (IL)-24 transforms the tumor microenvironment and induces anticancer immunity in a murine model of colon cancer.

Interleukin-24 (IL-24) is a novel tumor suppressor and can mediate the induction of Th1-type cytokines from peripheral blood mononuclear cells. The individual properties of IL-24 have been previously examined; however, its in vivo immunological consequences and antitumor properties have not been previously evaluated with respect to colon cancer, the most commonly diagnosed cancer in China. Thus, we evaluated whether IL-24 could inhibit the progression of colon cancer in murine models with intact immune competence and explored the mechanisms underlying the immunological effects of IL-24 on colon cancer progression in vivo. In these murine models, we found that IL-24 promoted CD4(+) T cells and CD8(+) T cells to secrete interferon gamma and enhanced the cytotoxicity of CD8(+) T cells in vivo. More importantly, we demonstrated that IL-24 transformed the tumor microenvironment and enhanced antitumor effects in favor of tumor eradication. Additionally, IL-24 expression correlated inversely with the clinical stage of human colorectal cancer. Thus, our study establishes a role of IL-24 in promoting antitumor immune responses and supports the development of a novel cytokine immunotherapy against colon cancer.

Targeting of interleukin (IL)-17A inhibits PDL1 expression in tumor cells and induces anticancer immunity in an estrogen receptor-negative murine model of breast cancer

The expression of IL-17A and programmed death ligand 1 (PDL1) is increased in estrogen receptor-negative breast cancer. IL-17A promotes tumor cell survival and invasiveness and inhibits the antitumor immune response. The PDL1–PD1 (programmed death protein 1) signaling pathway promotes escape from immune surveillance in tumor cells. The pro-tumor properties of IL-17A and PDL1 in various cancers have been previously examined; however, the relationship and roles of IL-17A and PDL1 in ER-negative breast cancer have not been evaluated. Therefore, we assessed whether IL-17A promotes PDL1 expression in tumor cells and whether targeting of IL-17A could inhibit ER-negative breast cancer progression in a murine model. Our study revealed that IL-17A promoted PDL1 expression in human and mouse cells. In the murine cancer model, targeting of IL-17A inhibited PDL1 expression in the tumor microenvironment, decreased the percentage of Treg cells in tumor-infiltrating lymphocytes, and promoted CD4+ and CD8+ T cells to secrete interferon gamma. More importantly, treatment with combined anti-IL-17A and anti-PDL1 antibodies enhanced antitumor effects in favor of tumor eradication. Thus, our study established a pro-tumor role of IL-17A in promoting tumor immune escape and supports the development of a novel cytokine immunotherapy against breast cancer.

Illegitimate RAG-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia.

Breakpoint cluster region‐Abelson murine leukaemia viral oncogene homologue 1 (BCR–ABL1), encoded by the Philadelphia (Ph) chromosome, is the characteristic of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). We demonstrated that expression of the Ik6 transcript, which lacked exons 3–6, was observed exclusively in BCR–ABL1+B ALL and lymphoid blast crisis CML (BC–CML) patients harbouring the IKZF1 Δ3–6 deletion. To confirm the hypothesis that illegitimate recombination activating gene protein (RAG)‐mediated recombination events are involved in IKZF1 Δ3–6 deletion in BCR–ABL1 lymphoblastic leukaemia, we first demonstrated that the expression rates of RAG1 and RAG2, collectively called RAG, were higher in ALL and BC–CML (lymphoid). Notably, analysis of relationships among RAG, BCR–ABL1 and Ikaros 6 (Ik6) showed that Ik6 can be generated only if RAG and BCR–ABL1 are co‐existing. The sequencing data showed that the deleted segments of introns 2 and 6 contained cryptic recombination signal sequences (cRSSs) and frequently had non‐template nucleotides inserted between breakpoints. Furthermore, we used chromatin immunoprecipitation (ChIP) technology and demonstrated that the sequences directly flanking IKZF1 Δ3–6 deletion breakpoints have significantly higher levels of histone H3 lysine 4 trimethylation (H3K4me3) modifications. Overall, RAG expression, good‐quality cRSS and a specific chromatin modification, H3K4me3, satisfy the conditions of RAGs off‐target effects on IKZF1. Our work provides evidence for RAG‐mediated IKZF1 Δ3–6 deletion. Our results raise the prospect that RAG is a valuable biomarker in disease surveillance. Dissecting the contribution of RAG should not only provide valuable mechanistic insights, but will also lead to a new therapeutic direction.

Association of serum interleukin‑10, interleukin‑17A and transforming growth factor‑α levels with human benign and malignant breast diseases

Interleukin-10 (IL-10), interleukin-17A (IL-17A) and transforming growth factor α (TGF-α) have been implicated in the progression of breast cancer. However, the diagnostic and prognostic roles of these cytokines in ductal carcinoma remain unclear. The present study therefore aimed to determine the serum levels of IL-10, IL-17 and TGF-α in subjects with benign and malignant breast diseases and to evaluate the clinical significance of these cytokines in ductal carcinoma. Pre-operative serum samples were collected from 378 patients with breast disease and 70 healthy subjects. IL-10, IL-17A and TGF-α levels were measured using ELISA. Serum levels of these cytokine in patients with different breast diseases were evaluated. Furthermore, correlations between levels of these cytokines and the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in ductal carcinoma were determined. The results demonstrated that serum levels of IL-10 and IL-17A were significantly increased in subjects with atypical hyperplasia and ductal carcinoma. Furthermore, IL-10 and IL-17A levels were increased in patients with a more serious clinical tumor stage and tumors that were ER- and PR-. Furthermore, high serum levels of TGF-α were associated with HER2+ tumors. A strong positive correlation was identified between TGF-α and IL-17A levels. Therefore, the results of the current study revealed that elevated serum IL-10, IL-17A and TGF-α levels are strongly associated with ductal carcinoma, specifically with tumor stage. High serum levels of IL-10 and IL-17A were also associated with the negative expression of ER and PR in ductal carcinoma, and high serum levels of TGF-α were associated with the positive expression of HER2 in ductal carcinoma. Thus, serum cytokine levels may be measured to identify patients with a poor prognosis who may benefit from more aggressive management and treatment.

AID and TET2 cooperation modulates FANCA expression by active demethylation in diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is traced to a mature B malignance carrying abnormal activation‐induced cytidine deaminase (AID) expression. AID activity initially focuses on deamination of cytidine to uracil to generate somatic hypermutation and class‐switch recombination of the immunoglobulin (Ig), but recently it has been implicated in DNA demethylation of genes required for B cell development and proliferation in the germinal centre (GC). However, whether AID activity on mutation or demethylation of genes involves oncogenesis of DLBCL has not been well characterized. Our data demonstrate that the proto‐oncogene Fanconi anaemia complementation group A (FANCA) is highly expressed in DLBCL patients and cell lines, respectively. AID recruits demethylation enzyme ten eleven translocation family member (TET2) to bind the FANCA promoter. As a result, FANCA is demethylated and its expression increases in DLBCL. On the basis of our findings, we have developed a new therapeutic strategy to significantly inhibit DLBCL cell growth by combination of the proteasome inhibitor bortezomib with AID and TET2 depletion. These findings support a novel mechanism that AID has a crucial role in active demethylation for oncogene activation in DLBCL.

RAG2 involves the Igκ locus demethylation during B cell development

HIGHLIGHTSWild‐type RAG2 involves the Ig&kgr; locus demethylation in a RAG1‐independent manner at pre‐B cell stage of B lymphocyte development.RAG2’s residues 350–383 region is responsible for endogenous Ig&kgr; locus demethylation.DNA methylation status does not guide the Ig&kgr; gene assembly in our working model. ABSTRACT The genes encoding the immunoglobulin &kgr; light chain are assembled during B cell development by V(D)J recombination. For efficient rearrangement, the Ig&kgr; locus must undergo a series of epigenetic changes. One such epigenetic mark is DNA methylation. The mechanism that the Ig&kgr; locus is selectively demethylated at the pre‐B cell stage has not previously been characterized. Here, we employed bisulfite DNA‐modification assays to analyze the methylation status of the Ig&kgr; locus in primary pre‐B cells from RAG‐deficient mice with pre‐rearranged Igh knock‐in allele. We observed that the Ig&kgr; locus was hypermethylated in RAG2‐deficient pre‐B cells but hypomethylated in RAG1‐deficient pre‐B cells, indicating that wild‐type (WT) RAG2 involves the Ig&kgr; locus demethylation in a RAG1‐independent manner prior to rearrangement. We generated a series of RAG2 mutants between residue 350 and 383. We showed that these mutants mediated the Ig&kgr; rearrangement but failed to regulate the Ig&kgr; gene demethylation. We further analyzed that these mutants could increase RAG recombinase activity in vivo. We conclude that residues 350–383 region are responsible for endogenous Ig&kgr; locus demethylation at pre‐B cells. We propose that WT RAG2 has an intrinsic function to regulate the Ig&kgr; locus demethylation.

Epigenetic modifications of the VH region after DJH recombination in Pro-B Cells

The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential DH–JH and VH–DJH recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of VH genes and the lack of a suitable model, the epigenetic modifications of VH genes after DJH recombination have not previously been characterized. Here, we employed two v‐Abl pro‐B cell lines, in which the Igh locus is in germline and DJH‐recombined configurations, respectively. The DJH junction displays the characteristics of a recombination centre, such as high levels of activation‐associated histone modifications and recombination‐activating gene protein (RAG) binding in DJH‐rearranged pro‐B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the VH region have distinct epigenetic characteristics after DJH recombination. Distal VH genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal VH genes are relatively close to the DJH recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJH recombination centre might serve as a cis‐acting element to regulate the accessibility of the VH region. Furthermore, we demonstrate that RAG weakly binds to functional VH genes, which is the first detailed assessment of RAG dynamic binding to VH genes. We provide a way for VH–DJH recombination in which the VH gene is brought into close proximity with the DJH recombination centre for RAG binding by a Pax5‐dependent chromosomal compaction event, and held in this position for subsequent cleavage and VH–DJH joining.