Programmed cell death ligand 1 (PD-L1) on tumor cells suppresses anti-tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD-L1 disruption in ovarian cancer. PD-L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. PD-L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD-L1-KO ID8-inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD-L1-KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4

BACKGROUND/AIM: Lung cancer is the leading cause of cancer-related death and a better marker for advanced personalized therapeutic approaches, such as immunotherapies, is in urgent need. Interleukin-12 (IL-12) is a cytokine that has been reported to exhibit potent tumoricidal effects, however, the contribution of IL-12 genotypes to lung cancer is still largely unrevealed. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in IL-12A and IL-12B are associated with lung cancer in a Taiwanese population.
MATERIALS AND METHODS: Genotypes of 358 lung cancer patients and 716 controls were determined by the polymerase chain reaction-restriction fragment length polymorphism method.
RESULTS: The distributions of genotypic (p=0.0036) and allelic (p=0.0005) frequencies of IL-12A rs568408 demonstrated significant differences between cases and controls. In detail, the AA genotype of IL-12A rs568408 was associated with a significantly elevated risk of lung cancer compared with the GG genotype (odds ratio(OR)=2.41, 95% confidence interval(CI)=1.36-4.29, p=0.0021). No difference was observed regarding IL-12A rs2243115 and IL-12B rs3212227 genotypes between the case and control groups. In addition, the results of interaction analysis showed that the AA genotype of IL-12A rs568408 was associated with elevated lung cancer risk, especially among those with smoking habits (p=0.0043).
CONCLUSION: IL-12A rs568404 AA genotype may contribute to the etiology and serve as a genomic determinant of lung cancer in Taiwanese, especially smokers.

Interleukin 35 (IL-35) is a potent immunosuppressive cytokine, consisting of an Epstein-Barr virus-induced gene 3 (EBI3) subunit and a p35 subunit. IL-35 is mainly produced by regulatory T and regulatory B cells, and plays a crucial role in the development and prevention of infectious and autoimmune diseases. However, the effect of IL-35 in malignant disease is not well understood. In this study, we demonstrated that breast cancer cells (BCCs) also expressed and secreted IL-35 and higher level of IL-35 in BCCs was closely associated with poor prognosis of patients and was an independent unfavorable prognostic factor for breast cancer. Subsequent study revealed that BCC-derived IL-35 inhibited conventional T (Tconv) cell proliferation and further induced suppressed Tconv cells into IL-35-producing induced regulatory T (iTr35) cells. Furthermore, BCC-derived IL-35 promoted the secretion of inhibitory cytokine IL-10 and obviously decreased the secretion of Th1-type cytokine IFN-γ and Th17-type cytokine IL-17 in Tconv cells. Meanwhile, the expression of inhibitory receptor CD73 was also elevated on the surface of Tconv cells following the BCCs' supernatant treatment. Mechanistically, BCC-derived IL-35 exhausted Tconv cells and induced iTr35 by activating transcription factor STAT1/STAT3. Hence, our results indicate functions of BCC-derived IL-35 in promoting tumor progression through proliferation inhibition of tumor-infiltrating Tconv cells and induction of iTr35 cells in tumor microenvironment. This study highlights that IL-35 produced by BCCs are a potential therapeutic target for breast cancer.

Rheumatoid arthritis patients often develop the diffuse large B-cell lymphoma subtype of methotrexate-associated lymphoproliferative disorder (DLBCL). We characterized the genomic profile and pathologic characteristics of 20 biopsies using an integrative approach. DLBCL was associated with extranodal involvement, a high/high-intermediate international prognostic index in 53% of cases, and responded to MTX withdrawal. The phenotype was nongerminal center B-cell in 85% of samples and Epstein-Barr encoding region positive (EBER) in 65%, with a high proliferation index and intermediate MYC expression levels. The immune microenvironment showed high numbers of CD8 cytotoxic T lymphocytes and CD163 M2 macrophages with an (CD163/CD68) M2 ratio of 3.6. Its genomic profile was characterized by 3p12.1-q25.31, 6p25.3, 8q23.1-q24.3, and 12p13.33-q24.33 gains, 6q22.31-q24.1 and 13q21.33-q34 losses, and 1p36.11-p35.3 copy neutral loss-of-heterozygosity. This profile was closer to nongerminal center B-cell DLBCL not-otherwise-specified, but with characteristic 3q, 12q, and 20p gains and lower 9p losses (P<0.05). We successfully verified array results using fluorescent DNA in situ hybridization on PLOD2, MYC, WNT1, and BCL2. Protein immunohistochemistry revealed that DLBCL expressed high IRF4 (6p25.3) and SELPLG (12q24.11) levels, intermediate TNFRSF14 (1p36.32; the exons 1 to 3 were unmutated), BTLA (3q13.2), PLOD2 (3q24), KLHL6 (3q27.1), and MYC (8q24.21) levels, and low AICDA (12p13.31) and EFNB2 (13q33.3) levels. The correlation between the DNA copy number and protein immunohistochemistry was confirmed for BTLA, PLOD2, and EFNB2. The characteristics of EBER versus EBER cases were similar, with the exception of specific changes: EBER cases had higher numbers of CD163 M2 macrophages and FOXP3 regulatory T lymphocytes, high programmed cell death 1 ligand 1 expression levels, slightly fewer genomic changes, and 3q and 4p focal gains. In conclusion, DLBCL has a characteristic genomic profile with 3q and 12 gains, 13q loss, different expression levels of relevant pathogenic biomarkers, and a microenvironment with high numbers of cytotoxic T lymphocytes and M2 macrophages.

BACKGROUND: CDK5RAP3 was initially isolated as a binding protein of the CDK5 activator p35. Although CDK5RAP3 has been shown to negatively regulate the Wnt/β-catenin signaling pathway in gastric cancer by repressing GSK-3β phosphorylation, its in-depth mechanism has not been determined.
METHODS: Following CDK5RAP3 overexpression or knock down, CDK5RAP3 signaling pathways were investigated in gastric cancer cells by Western Blotting. Cell growth, invasion and migration were also evaluated in gastric cancer cell lines. We analyzed CDK5RAP3, AKT, p-AKT (Ser473), GSK-3β and p-GSK-3β (Ser9) expression in gastric tumor samples and adjacent non-tumor tissues from 295 patients using immunohistochemistry and Western Blotting. The prognostic significance of CDK5RAP3 and p-AKT (Ser473) was confirmed by a Log-rank test.
RESULTS: Our study demonstrated that the expression of p-AKT (Ser473) and p-GSK-3β (Ser9) was negatively correlated with CDK5RAP3 in stable gastric cancer cell lines. CDK5RAP3 repressed AKT phosphorylation, which promoted GSK-3β phosphorylation, thereby suppressing β-catenin protein expression and, consequently, gastric cancer. The protein level of CDK5RAP3 was markedly decreased in most gastric tumor tissues compared with adjacent non-tumor tissues, and the levels of p-AKT (Ser473) and p-GSK-3β (Ser9) were also negatively correlated with those of CDK5RAP3. The prognostic value of CDK5RAP3 for overall survival was found to be dependent on AKT phosphorylation.
CONCLUSION: Our results demonstrated that CDK5RAP3 negatively regulates the Wnt/β-catenin signaling pathway by repressing AKT phosphorylation, which leads to better survival of patients with gastric cancer.

Tumor metastasis depends on the dynamic regulation of cell adhesion through β1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFβ1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of β1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of β1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates β1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells.

Cancer cells are adept at evading cell death, but the underlying mechanisms are poorly understood. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is composed of two different subunits, p40 and p35. This study underlines the importance of IL-12 p40 monomer (p40) in helping cancer cells to escape cell death. We found that different mouse and human cancer cells produced greater levels of p40 than p40 homodimer (p40

BACKGROUND & AIMS: Cells of the monocyte lineage contribute to tumor angiogenesis. Interleukin 35 (IL35) is a member of the IL12 family produced by regulatory, but not effector, T cells. IL35 is a dimer comprising the IL12 alpha and IL27 beta chains, encoded by IL12A and EBI3, respectively. Expression of IL35 is increased in pancreatic ductal adenocarcinomas (PDACs) compared with normal pancreatic tissues, and promotes metastasis. We investigated the role of IL35 in monocyte-induced angiogenesis of PDAC in mice.
METHODS: We measured levels of IL35 protein, microvessel density, and numbers of monocytes in 123 sequential PDAC tissues from patients who underwent surgery in China in 2010. We performed studies with the human PDAC cell lines CFPAC-1, BxPC-3, Panc-1, MIA-PaCa-2, and mouse PDAC cell line Pan02. Monocyte subsets were isolated by flow cytometry from human peripheral blood mononuclear cells. Fused human or mouse IL12A and EBI3 genes were overexpressed in PDAC cells or knocked down using small hairpin RNAs. Cells were grown as xenograft tumors in SCID mice; some mice were given injections of an IL35-neutralizing antibody and tumor growth was monitored. We performed chemotaxis assays to measure the ability of IL35 to recruit monocytes. We analyzed mRNA sequences of 179 PDACs in the Cancer Genome Atlas to identify correlations between expression of IL12A and EBI3 and monocyte markers. Monocytes incubated with IL35 or PDAC cell supernatants were analyzed in tube formation and endothelial migration assays.
RESULTS: In PDAC samples from patients, levels of IL35 mRNA and protein correlated with microvessel density and infiltration of monocyte lineage cells. In cells and mice with xenograft tumors, IL35 increased recruitment of monocytes into PDAC tumors, which required CCL5. Upon exposure to IL35, monocytes increased expression of genes whose products promote angiogenesis (CXCL1 and CXCL8). IL35 activated transcription of CCL5, CXCL1, and CXCL8 by inducing GP130 signaling, via IL12RB2 and phosphorylation of STAT1 and STAT4. A combination of a neutralizing antibody against IL35 and gemcitabine significantly decreased monocyte infiltration, microvessel density, and volume of xenograft tumors grown from PDAC cells in mice.
CONCLUSIONS: PDAC cells produce IL35 to recruit monocytes via CCL5 and induce macrophage to promote angiogenesis via expression of CXCL1 and CXCL8. IL35 signaling promotes angiogenesis and growth of xenograft tumors from PDAC cells in mice. IL35 might serve as a therapeutic target for patients with pancreatic cancer.

There is a dearth of knowledge about the pathogenesis of premalignant lung lesions, especially for atypical adenomatous hyperplasia (AAH), the only known precursor for the major lung cancer subtype adenocarcinoma (LUAD). In this study, we performed deep DNA and RNA sequencing analyses of a set of AAH, LUAD, and normal tissues. Somatic

OBJECTIVE: Lung cancer, particularly the non-small-cell lung cancer (NSCLC) subtype, is the leading cause of cancer-related death worldwide. Several functional polymorphisms in inflammatory cytokine genes, such as IL1B, IL6, IL12A, IL13 and IL16, have been associated with the risk of NSCLC. The aim of this study was to evaluate the association between ILs gene polymorphisms and the risk of developing NSCLC.
PARTICIPANTS AND METHODS: A retrospective case-control study was carried out, including 174 NSCLC cases and 298 controls of Spanish origin. IL1B (rs1143634), IL1B (rs12621220), IL1B (rs1143623), IL1B (rs16944), IL1B (rs1143627), IL12A (rs662959), IL13 (rs1881457), IL6 (rs1800795) and IL16 (rs7170924) gene polymorphisms were analysed by TaqMan.
RESULTS: The genotypic logistic regression model adjusted by smoking status showed that the IL1B rs1143634-TT genotype was associated with a lower risk of NSCLC (P=0.04312; odds ratio=0.226; 95% confidence interval=0.044-0.840). No other gene polymorphisms showed an association with NSCLC in any of the models tested.
CONCLUSION: In conclusion, IL1B rs1143634 was significantly associated with a higher risk of NSCLC. No influence of IL1B rs12621220, rs1143623, rs16944, rs1143627, IL12A rs662959, IL13 rs1881457 and IL16 rs7170924 on the risk of developing NSCLC was found in our study.

BACKGROUND: Surgery is the standard treatment for early-stage NSCLC, and platinum-based chemotherapy remains as the treatment of choice for advanced-stage NSCLC patients with naïve EGFR status. However, overall 5-years relative survival rates are low. Interleukins (ILs) are crucial for processes associated with tumor development. In NSCLC, IL1B, IL6, IL12A, IL13 and IL16 gene polymorphisms may contribute to individual variation in terms of patient survival. The purpose of this study was to evaluate the association between IL gene polymorphisms and survival in NSCLC patients.
METHODS: A prospective cohorts study was performed, including 170 NSCLC patients (114 Stage IIIB-IV, 56 Stage I-IIIA). IL1B (C > T; rs1143634), IL1B (C > T; rs12621220), IL1B (C > G; rs1143623), IL1B (A > G; rs16944), IL1B (C > T; rs1143627), IL6 (C > G; rs1800795), IL12A (C > T; rs662959), IL13 (A > C; rs1881457) and IL16 (G > T; rs7170924) gene polymorphisms were analyzed by PCR Real-Time.
RESULTS: Patients with IL16 rs7170924-GG genotype were in higher risk of death (p = 0.0139; HR = 1.82; CI
CONCLUSIONS: Our results suggest that IL16 rs7170924-GG and IL12A rs662959-TT genotypes predict higher risk of death and progression, respectively, in NSCLC patients. No influence of IL1B rs12621220, IL1B rs1143623, IL1B rs16944, IL1B rs1143627, IL6 rs1800795, IL13 rs1881457 on NSCLC clinical outcomes was found in our patients.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma. Despite a favorable therapeutic response to first-line chemo-immunotherapy, still 30-40% of patients is refractory, or relapse after this treatment. Thus, alternative strategies must be sought. Previous studies have indicated that cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase, is involved in tumor development and progression, and it may represent a potential therapeutic target. However, its role in modulating DLBCL growth and progression remains largely unexplored. In this study, we show that CDK5 and its activator, cyclin-dependent kinase 5 activator 1 (CDK5R1 or p35), are overexpressed in DLBCL cell lines and that signal transducer and activator of transcription 3 (STAT3) phosphorylation and activity is dependent on CDK5 expression in DLBCL. Using public data sets, we also demonstrate that patients with DLBCL show a higher expression of CDK5 compared with healthy individuals. By using loss-of-function approaches, we demonstrate that CDK5's activity regulates proliferation and survival of DLBCL cells. MicroRNAs (miRNAs or miRs) are small noncoding RNAs that negatively regulating gene expression and are involved in cancer initiation and progression. We identify miR-26a as direct regulator of p35 expression and CDK5 activity. We show that miR-26a expression is lower in DLBCL cell lines compared to B lymphocytes and that its ectopic expression leads to a drastic reduction of DLBCL tumor growth in vivo and decreased proliferation, cell-cycle progression, and survival in vitro. Remarkably, concomitant overexpression of a 3'-UTR-truncated form of p35 promoted tumor growth in vivo and cell proliferation, cell-cycle progression, and cell survival in vitro. In conclusion, these results demonstrate an important role for miR-26a and CDK5 together in the survival and growth of DLBCL cells, suggesting the existence of potential novel therapeutic targets for the treatment of DLBCL.

Interleukin- (IL-) 35 is a member of the IL-12 cytokine family and a heterodimeric protein formed by Epstein-Barr-induced gene 3 (EBI3) and IL-12p35. Emerging evidence shows that IL-35 is a key player in the regulation of cellular communication, differentiation, and inflammation. Altered IL-35 expression has been found in disease conditions such as cancer, rheumatoid arthritis, and, more recently, asthma. In cancer, IL-35 is involved in the regulation of tumorigenesis, cancer progression, and metastasis. In rheumatoid arthritis, IL-35 acts as a negative regulator of inflammation. Similarly, IL-35 also appears to suppress allergic inflammation in asthma. In an in vivo murine model of asthma, transfer of adenovirus-mediated IL-35 markedly reduced the degree of airway hyperresponsiveness (AHR) and inflammatory cell infiltration. Many studies have shown the involvement of IL-35 in a number of aspects of allergic inflammation, such as eosinophil and neutrophil recruitment as well as inhibition of inflammatory mediators of the Th2 subtype. However, the exact molecular mechanisms underlying the role of IL-35 in human asthma have yet to be fully elucidated. This review describes the current evidence regarding the role of IL-35 in the pathophysiology of asthma and evaluates the potential of IL-35 as a biomarker for airway inflammation and a therapeutic target for the treatment of asthma.

Many molecular epidemiologic studies have explored the possible links between interleukin-12 (IL-12) polymorphisms and various cancers. However, results from these studies remain inconsistent. This meta-analysis is aimed to shed light on the associations between three common loci (rs568408, rs2243115, rs3212227) of IL-12 gene and overall cancer risk. Our meta-analysis finally included 33 studies comprising 10,587 cancer cases and 12,040 cancer-free controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the cancer risk. We observed a significant association between IL-12B rs3212227 and overall cancer risk, especially in hepatocellular carcinoma, nasopharyngeal cancer, and among Asians. IL-12A polymorphisms (rs2243115 and rs568408) were found no influence on overall cancer risk. Nevertheless, stratification analyses demonstrated that rs568408 polymorphism contributed to increasing cancer risk of Caucasians and cervical cancer. And, rs2243115 may enhance the risk of brain tumor. These findings provided evidence that IL-12 polymorphisms may play a potential role in cancer risk.

The associations between interleukin-12 (IL-12) gene polymorphisms and cancer risk have been discussed extensively, with controversial results. Therefore, we conducted the present meta-analysis to better assess the potential roles of IL-12 gene variation in cancer occurrence. Eligible articles were found via PubMed, Medline, EMBASE, Google Scholar and CNKI. Odds ratios and 95% confidence intervals were used to evaluate the associations between IL-12 gene polymorphisms and cancer risk. Thirty-one studies with 10,749 cancer patients and 11,921 healthy subjects were included in the analyses. The overall results showed that cancer risk was increased by IL-12A rs568408 (GG versus GA + AA: P = 0.004; G versus A: P = 0.005) and IL-12B rs3212227 (AA versus AC + CC: P = 0.004; CC versus AA + AC: P = 0.03; A versus C: P = 0.007) polymorphisms. Further subgroup analyses for IL-12A rs568408 and IL-12B rs3212227 revealed that the positive results could be impacted by the ethnicity of the population, cancer type and/or genotyping methods. However, we failed to detect any significant associations between the IL-12A rs2243115 polymorphism and cancer risk in either the overall or the subgroup analyses. The current study suggests that certain IL-12 gene polymorphisms serve as biological markers of cancer susceptibility.

As an atypical member of cyclin dependent kinase family, Cyclin dependent kinase 5 (Cdk5) is considered as a neuron-specific kinase in the past decade due to the abundant existence of its activator p35 in post-mitotic neurons. Recent studies show that Cdk5 participates in a series of biological and pathological processes in non-neuronal cells, and is generally dysregulated in various cancer cells. The inhibition or knockdown of Cdk5 has been proven to play an anti-cancer role through various mechanisms, and can synergize the killing effect of chemotherapeutics. DNA damage response (DDR) is a series of regulatory events including DNA damage, cell-cycle arrest, regulation of DNA replication, and repair or bypass of DNA damage to ensure the maintenance of genomic stability and cell viability. Here we describe the regulatory mechanisms of Cdk5, its controversial roles in apoptosis and focus on its links to DDR and cancer.

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1-fibrinogen-ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression.

BACKGROUND: Hepatocellular carcinoma has been recorded the commonest cancer in Egypt. This increasing incidence may be attributed to the high prevalence of hepatitis C virus with its complications, so this study aimed to investigate the association between the potentially functional polymorphisms of IL12A and IL12B genes as a risk factor of development of hepatocellular carcinoma (HCC) on top of hepatitis C virus (HCV) infection in an Egyptian population.
MATERIALS AND METHODS: We genotyped two loci of IL12 which were rs568408 (3'UTR G>A) for IL12A and rs3212227 (3'UTR A>C) for IL12B in 78 patients with HCC on top of chronic HCV infection. In addition, 64 cancer-free chronic HCV patients were studied, besides 92 healthy subjects who were included as control.
RESULTS: Study of rs568408 (G>A) gene polymorphism showed that the A allele is higher while the G allele is lower in HCC cases than cancer-free chronic HCV patients (p = 0.006*). The A-containing genotypes AG and (AG+AA) were higher while the GG was lower (p = 0.009* and p = 0.005*), respectively. The study of the rs3212227 (A>C) polymorphism showed neither statistically significant differences between the C and A allele (p = 0.2) nor between CC, AC, or AC+CC in HCC cases and cancer-free chronic HCV patients (p = 0.7, p = 0.2, and p = 0.29), respectively.
CONCLUSION: Our findings showed that IL12A rs568408 (G>A) polymorphism may contribute to the risk of HCC on top of chronic HCV infection, whereas that of IL12B rs3212227 (A>C) do not.

CDK5RAP3 was isolated as a binding protein of the Cdk5 activator p35. Although CDK5RAP3 has been implicated in cancer progression, its expression and function have not been investigated in gastric cancer. Our study demonstrated that the mRNA and protein levels of CDK5RAP3 were markedly decreased in gastric tumor tissues when compared with respective adjacent non-tumor tissues. CDK5RAP3 in gastric cancer cells significantly reduced cell proliferation, migration, invasion and tumor xenograft growth through inhibition of β-catenin. Secondly, CDK5RAP3 was found to suppress the phosphorylation of GSK-3β (Ser9), leading to the phosphorylation (Ser37/Thr41) and subsequent degradation of β-catenin. Lastly, the prognostic value of CDK5RAP3 for overall survival was found to be dependent on β-catenin cytoplasm/nucleus localization in human gastric cancer samples. Collectively, our results demonstrated that CDK5RAP3 negatively regulates the β-catenin signaling pathway by repressing GSK-3β phosphorylation and could be a potential therapeutic target for gastric cancer.

IL-35 is a novel heterodimeric and inhibitory cytokine, composed of interleukin-12 subunit alpha (P35) and Epstein-Barr virus -induced gene 3 (EBI3). IL-35 has been reported to be produced by a range of cell types, especially regulatory T cells, and to exert immunosuppressive effects via the STATx signaling pathway. In this study, we demonstrated that IL-35 expression was elevated in both serum and tumors in patients with colorectal cancer. IL-35 mainly expressed in CD4+ T cells in human colorectal cancer tumors and adjacent tissues. Increased IL-35 expression in tumor-adjacent tissues was significantly associated with tumor metastasis. IL-35 inhibited the proliferation of CD4+CD25- T effector cells in vitro in a dose-dependent manner, and its suppression was partially reversed by applying IL-35-neutralizing antibodies. IL-35 treatment activated the phosphorylation of both STAT1 and STAT3 in human CD4+ T cells. Meanwhile, IL-35 induced a positive feedback loop to promote its own production. We observed that Tregs obtained from colorectal cancer patients were capable of inducing more IL-35 production. In addition, EBI3 promoter-driven luciferase activity was higher than that of the mock plasmid after IL-35stimulation. Thus, our study indicates that the high level of IL-35 in colorectal cancer promotes the production of IL-35 via STAT1 and STAT3, which suppresses T cell proliferation and may participate in tumor immunotolerance.

Data on interleukin-31 (IL-31) involvement in the patho-genesis of mastocytosis, and its impact on pruritus development in the disease, are limited. The aim of this study was to analyse distinct IL-31 gene polymorphisms in 127 patients (age 0.5-76 years) with mastocytosis and their correlation with clinical presentation, pruritus and serum IL-31 levels. In patients with mastocytosis, the frequency of IL-31 IVS2+12AA genotype and IVS2+12A allele was higher than in control subjects and they were linked to an increased risk of development of mastocytosis. In adult patients, but not in children, -2057AA genotype was also associated with an increased risk of occurrence of mastocytosis. Pruritus affected 83.3% of 78 adult patients with mastocytosis, and a positive correlation between serum IL-31 levels and pruritus was found in these patients. In conclusion, distinct polymorphic variants of the IL-31 gene may be involved in the patho-genesis of mastocytosis, and IL-31 may be involved in the induction of pruritus in patients with mastocytosis.

Glioblastoma (GBM) is the most common primary brain tumor, with poor prognosis and a lack of effective therapeutic options. The aberrant expression of transcription factor REST (repressor element 1-silencing transcription factor) had been reported in different kinds of tumors. However, the function of REST and its mechanisms in GBM remain elusive. Here, REST expression was inhibited by siRNA silencing in U-87 and U-251 GBM cells. Then CCK-8 assay showed significantly decreased cell proliferation, and the inhibition of migration was verified by scratch wound healing assay and transwell assay. Using cell cycle analysis and Annexin V/PI straining assay, G1 phase cell cycle arrest was found to be a reason for the suppression of cell proliferation and migration upon REST silencing, while apoptosis was not affected by REST silencing. Further, the detection of REST-downstream genes involved in cytostasis and migration inhibition demonstrated that CCND1 and CCNE1 were reduced; CDK5R1, BBC3, EGR1, SLC25A4, PDCD7, MAPK11, MAPK12, FADD and DAXX were enhanced, among which BBC3 and DAXX were direct targets of REST, as verified by ChIP (chromatin immunoprecipitation) and Western blotting. These data suggested that REST is a master regulator that maintains GBM cells proliferation and migration, partly through regulating cell cycle by repressing downstream genes, which might represent a potential target for GBM therapy.

Gender disparity has long been considered as a key to fully understand hepatocellular carcinoma (HCC) development. At the same time, immunotherapy related to IL12 still need more investigation before being applied in clinical settings. The aim of this study is to investigate the influence of the androgen receptor (AR) on natural killer (NK) cell-related innate immune surveillance in liver cancer, and provide a novel therapeutic approach to suppress HCC via altering IL12A. By using in vitro cell cytotoxicity test and in vivo liver orthotopic xenograft mouse model, we identified the role of AR in modulating NK cell cytotoxicity. Luciferase report assay and chromatin immunoprecipitation assay were applied for mechanism dissection. IHC was performed for sample staining. Our results showed AR could suppress IL12A expression at the transcriptional level via direct binding to the IL12A promoter region that resulted in repressing efficacy of NK cell cytotoxicity against HCC, and sorafenib treatment could enhance IL12A signals via suppressing AR signals. These results not only help to explain the AR roles in the gender disparity of HCC but also provide a potential new therapy to better suppress HCC via combining sorafenib with NK cell-related immunotherapy. Mol Cancer Ther; 15(4); 731-42. ©2016 AACR.

BACKGROUND: Abnormal activation of STAT3 and miR-21 plays a vital role in progression and invasion of solid tumors. The cyclin-dependent kinase 5 (CDK5) is reported to contribute to cancer metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of STAT3/miR-21 axis and CDK5 in head and neck squamous cell carcinoma remains unclear.
METHODS: We measured the expression of miR-21, CDK5 and EMT markers in 60 HNSCC tumor samples. We used Immunohistochemistry and in situ hybridization assay to examine the role of STAT3/miR-21 axis and CDK5 activation in the invasiveness of HNSCC. The clinical survival relevance was analyzed by Kaplan-Meier analysis and univariate/multivariate COX regression model. Multiple approaches including scratch, transwell chamber assay and other molecular biology techniques were used to validate the anti-invasion effect of targeting miR-21 in Tca8113 and Hep-2 cell lines in vitro. Furthermore, whether miR-21 depletion inhibits HNSCC invasion in vivo was confirmed in Tca8113 xenograft tumor model.
RESULTS: The expression of miR-21 and CDK5 were significantly correlated with lymph node metastasis in HNSCC. Hep-2 and Tca8113 cell lines showed co-overexpression of miR-21 and CDK5. WP1066 or asON-miR-21 treatment depleted miR-21 and CDK5 expression and significantly inhibited migration or invasion in Hep-2 and Tca8113 cells. The expression levels of CDK5/p35, N-cadherin, vimentin, β-catenin were inhibited while E-cadherin level was increased by miR-21 depletion in vitro and in vivo. Conversely, ectopic CDK5 overexpression significantly induced tumor cell motility and EMT. Moreover, ectopic CDK5 overexpression in Hep-2 and Tca8113 cells rescued the observed phenotype after miR-21 silencing or WP1066 treatment.
CONCLUSIONS: miR-21 cooperates with CDK5 to promote EMT and invasion in HNSCC. This finding suggests that CDK5 may be an important cofactor for targeting when designing metastasis-blocking therapy by targeting STAT3/miR-21 axis with STAT3 inhibitor or miR-21 antisense oligonucleotide. This is the first demonstration of the novel role of STAT3/miR-21 axis and CDK5/CDK5R1 (p35) in metastasis of HNSCC.

IL12 plays a major role not only in inducing appropriate immune responses against viral infections (including HBV) but also in the antitumor immune response. This study was conducted to investigate the relationships of genetic variants in IL12 with hepatitis B virus (HBV) clearance and development of HBV-related hepatocellular carcinoma (HCC). We genotyped three single nucleotide polymorphisms (SNPs) of the IL12A (rs568406 and rs2243115) and IL12B (rs3212227) in 395 HBV-positive HCC patients, 293 persistent HBV carriers and 686 subjects with HBV natural clearance from southern China, using the improved multiplex ligase detection reaction (iMLDR) method. Logistic regression analysis adjusted for age, smoking, and alcohol consumption status showed that rs568408 variant genotypes were significantly associated with host HBV-related HCC risk when compared with persistent HBV carriers, and carriers of the GA + AA genotype decreased the HCC risk in comparison with GG carriers (adjusted OR = 0.53, 95 % CI 0.35-0.80, P = 0.002). No relationships between the rs2243115 and rs3212227 SNPs and HCC risk were observed (all P > 0.05). Besides, rs568408 showed an approaching significant effect on susceptibility to HBV persistent infection (adjusted OR = 1.34, 95 % CI 0.99-1.81, P = 0.057 in dominant genetic models). Furthermore, the TG haplotype was observed to be associated with a significantly increased risk of HBV-related HCC (OR = 1.42, 95 % CI 1.10-1.83, P = 0.006), while TA haplotype was associated with a decreased risk of HBV-related HCC (OR = 0.61, 95 % CI 0.45-0.83, P = 0.002). Our results reveal that the IL12A rs568408 variant may be a marker SNP for risk of both HBV clearance and HBV-related HCC development.

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

BACKGROUND: Malignant B-cell clones are affected by both acquired genetic alterations and by inherited genetic variations changing the inflammatory tumour microenvironment.
METHODS: We investigated 50 inflammatory response gene polymorphisms in 355 B-cell non-Hodgkin's lymphoma (B-NHL) samples encompassing 216 diffuse large B cell lymphoma (DLBCL) and 139 follicular lymphoma (FL) and 307 controls. The effect of single genes and haplotypes were investigated and gene-expression analysis was applied for selected genes. Since interaction between risk genes can have a large impact on phenotype, two-way gene-gene interaction analysis was included.
RESULTS: We found inherited SNPs in genes critical for inflammatory pathways; TLR9, IL4, TAP2, IL2RA, FCGR2A, TNFA, IL10RB, GALNT12, IL12A and IL1B were significantly associated with disease risk and SELE, IL1RN, TNFA, TAP2, MBL2, IL5, CX3CR1, CHI3L1 and IL12A were, associated with overall survival (OS) in specific diagnostic entities of B-NHL. We discovered noteworthy interactions between DLBCL risk alleles on IL10 and IL4RA and FL risk alleles on IL4RA and IL4. In relation to OS, a highly significant interaction was observed in DLBCL for IL4RA (rs1805010) * IL10 (rs1800890) (HR = 0.11 (0.02-0.50)). Finally, we explored the expression of risk genes from the gene-gene interaction analysis in normal B-cell subtypes showing a different expression of IL4RA, IL10, IL10RB genes supporting a pathogenetic effect of these interactions in the germinal center.
CONCLUSIONS: The present findings support the importance of inflammatory genes in B-cell lymphomas. We found association between polymorphic sites in inflammatory response genes and risk as well as outcome in B-NHL and suggest an effect of gene-gene interactions during the stepwise oncogenesis.

Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, but the etiology of it remains poorly understood. IL-35 is a recently described cytokine composed of an IL-12 subunit p35 and an IL-27 subunit Epstein-Barr virus induced gene 3 (EBI3), and has an immunosuppressive effect on inflammation through induction of regulatory T cells (Tregs) and suppression of Th1 and Th17. Recently, we have illustrated that concentrations of IL-35 in peripheral blood are up-regulated in newly diagnosed (ND) AML patients. However, whether IL-35 in bone marrow is increased in AML patients is not clear. In this study, we examined IL-35 in bone marrow by various methods including RT-PCR, ELISA, FCM and IHC, and found that IL-35 levels are also increased significantly in bone marrow of adult AML patients. Furthermore, we investigated that concentrations of bone marrow IL-35 in ND group were higher than that in complete remission (CR) group and control group, but there was no significant difference compared to that in relapse group. In conclusion, IL-35 was elevated in bone marrow of adult AML patients and this increase was correlated with the clinical stages of malignancy, suggesting that IL-35 is involved in pathogenesis of AML.