The interleukin (IL)-6/glycoprotein (GP)130/signal transducer and activator of transcription (STAT)3 pathway is emerging as a target for the treatment of hepatocellular carcinoma. IL-6 binds to IL-6R, forming a binary complex, which further combines with GP130 to transduce extracellular signaling by activating STAT3. Therefore, blocking the interaction between IL-6 and GP130 may inhibit the IL-6/GP130/STAT3 signaling pathway and its biological effects. It has been reported that bazedoxifene acetate (BAZ), a selective estrogen receptor modulator approved by the US Food and Drug Administration, could inhibit IL-6/GP130 protein-protein interactions. Western blot, immunofluorescence staining, wound healing and colony formation assays were used to detect the effect of BAZ on liver cancer cells. Cell viability was evaluated by MTT assay. Apoptosis of cells was determined using the Annexin V-FITC detection kit. Mouse xenograft tumor models were utilized to evaluate the effect of BAZ in vivo. Our data showed that BAZ inhibited STAT3 phosphorylation (P-STAT3) and expression of STAT3 downstream genes, inducing apoptosis in liver cancer cells. BAZ inhibited P-STAT3 induced by IL-6, but not by leukemia inhibitory factor. BAZ inhibited P-STAT1 and P-STAT6 less significantly as elicited by interferon-α, interferon-γ and IL-4. In addition, pretreatment of BAZ impeded the translocation of STAT3 to nuclei induced by IL-6. BAZ inhibited cell viability, wound healing and colony formation in vitro. Furthermore, tumor growth in HEPG2 mouse xenografts were significantly inhibited by daily intragastric gavage of BAZ. Our results suggest that BAZ inhibited the growth of hepatocellular carcinoma in vitro and in vivo, indicating another potential strategy for HCC prevention and therapy.

Endometrial cancer is common among postmenopausal women and its incidence is increasing in developed countries. Considering that >80% of endometrial cancers are assumed to be estrogen-related, higher estrogen exposure will be relevant to tumorigenesis. Therefore, the roles of estrogen target genes will be important to understand the pathophysiological mechanisms. We previously revealed that estrogen-responsive RING finger protein Efp contributes to breast cancer progression through the protein degradation of cell cycle checkpoint 14-3-3σ. We and others also proposed that Efp has tumor-promoting activities in estrogen receptor (ER)-negative cancer cells. In addition, Efp plays a role in type I interferon production by activating antiviral signaling, which provokes nuclear factor-κB (NF-κB) signaling. In the present study, we investigate whether Efp plays a critical role in endometrial cancer biology. We show that siRNA-mediated Efp knockdown represses the proliferation and migration of endometrial cancer ER-positive Ishikawa and ER-negative HEC-1A cells. Efp knockdown increases 14-3-3σ protein levels and decreases the rates proliferative stage cells. Efp siRNA significantly inhibits the in vivo tumor growth of endometrial cancer cells in both subcutaneous and orthotopic xenograft models. Intriguingly, Efp knockdown represses NF-κB-dependent transactivation and transcription of target genes, such as IL6ST and IL18, in endometrial cancer cells. Overall, Efp would exert a tumor-promoting role through modulating NF-κB pathway and 14-3-3σ protein degradation in endometrial cancer regardless of its estrogen receptor status. Our results indicate that Efp could be a potential diagnostic and therapeutic target for endometrial cancer.

BACKGROUND & AIMS: Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice.
METHODS: We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan.
RESULTS: We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130
CONCLUSIONS: We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.

BACKGROUND: The activation of multiple signaling pathways jeopardizes the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutation positive non-small cell lung cancer (NSCLC). Integrin-linked kinase (ILK) regulates the interactions between tumor cells and extracellular environment to activate signaling pathways and promote cell proliferation, migration, and epithelial-mesenchymal transition. Src homology 2 domain-containing phosphatase 2 (SHP2) is essential for receptor tyrosine kinase signaling and mitogen-activated protein kinase (MAPK) pathway activation.
METHODS: We analyzed tumor ILK, β-receptor subunit glycoprotein 130 (gp130), SHP2, and stromal hepatocyte growth factor (HGF) and interleukin-6 (IL-6) mRNA expression in baseline tumor specimens of advanced EGFR-mutation positive NSCLC patients treated with EGFR TKIs.
RESULTS: ILK, when highly expressed, was an independent poor prognostic factor for the progression-free survival of the patients, both in the univariate (hazard ratio [HR for disease progression, 2.49; 95% CI, 1.37-4.52; P = .0020]) and in the multivariate (HR 3.74; 95% CI, 1.33-10.56; P = .0126) Cox regression model. Patients with high SHP2 expression had an almost 13-month shorter progression-free survival (P = .0094) and an 18-month shorter overall survival (P = .0182) in comparison to those with low SHP2 mRNA expression.
INTERPRETATION: The levels of ILK and SHP2 could be predictive for upfront combinatory therapy of EGFR TKIs plus SHP2 or ILK inhibitors. FUND: A grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), an Instituto de Salud Carlos III grant (PI14/01678), a Marie Skłodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE).

STAT3 is a transcription factor that plays central roles in various physiological processes and its deregulation results in serious diseases including cancer. The mechanisms on how STAT3 activity is regulated remains enigmatic. Here we identify TRIM27 as a positive regulator of II-6-induced STAT3 activation and downstream gene expression. TRIM27 localizes to retromer-positive punctate structures and serves as a critical link for recruiting gp130, JAK1, and STAT3 to and subsequent phosphorylation of STAT3 at the retromer-positive structures. Overexpression of TRIM27 promotes cancer cell growth in vitro and tumor growth in nude mice, whereas knockdown of TRIM27 has opposite effects. Deficiency of TRIM27 significantly impairs dextran sulfate sodium (DSS)-induced STAT3 activation, inflammatory cytokine expression and colitis as well as azoxymethane (AOM)/DSS-induced colitis-associated cancer in mice. These findings reveal a retromer-dependent mechanism for regulation of STAT3 activation, inflammation, and inflammation-associated cancer development.

The RNA-binding protein Musashi 2 (MSI2) has emerged as an important regulator in cancer initiation, progression, and drug resistance. Translocations and deregulation of the

Neoadjuvant chemoradiotherapy (nCRT) following surgery significantly improves the survival rate of patients with rectal cancer. However, nCRT is associated with significant adverse symptoms and high medical costs. Therefore, it is important to investigate potential biomarkers for the prediction of the response to nCRT in patients with rectal cancer. The present study identified candidate biomarkers for predicting a complete response (CR) to nCRT in patients with rectal cancer and investigated the associated mechanisms. Microarray data (accession no. GSE29298) was downloaded from the Gene Expression Omnibus database. Differentially expressed microRNAs (miRNAs/miR) were screened between the pathological CR (pCR) group and no pCR (incomplete response) group. miRNA target genes were predicted using the miRWalk 2.0 online tool and subjected to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Furthermore, a miRNA co‑regulatory network was constructed and disease‑associated genes were predicted. The results demonstrated that a total of 36 upregulated and 5 downregulated miRNAs were identified between the two groups. Among these differentially expressed miRNAs, miR‑548c‑5p, miR‑548d‑5p and miR‑663a were significantly associated with a CR to nCRT. The co‑regulatory network and pathway analysis indicated that miR‑548c‑5p and miR‑548d‑5p may function together through stem cell pluripotency and ubiquitin‑mediated proteolysis signaling pathways. Furthermore, the prediction of disease‑associated genes demonstrated that miR‑548c‑5p/miR‑548d‑5p and miR‑663a may regulate genes associated with rectal cancer, including mutated in colorectal cancers (MCC) and adenomatous polyposis coli (APC), and colorectal neoplasms, including interleukin‑6 signal transducer (IL6ST), cell cycle checkpoint kinase 2 (CHEK2), marker of proliferation Ki‑67 (MKI67), cadherin 7 (CDH7), calreticulin (CALR) and transforming growth factor β1 (TGFB1). Therefore, miR‑548c‑5p, miR‑548d‑5p and miR‑663a are promising candidate biomarkers for predicting a CR to nCRT. miR‑548c‑5p/miR‑548d‑5p may be associated with a CR by regulating IL6ST, CHEK2, MKI67 and MCC. In addition, it may function through the pluripotency of stem cells and ubiquitin‑mediated proteolysis signaling pathways. miR‑663a may be associated with a CR to nCRT by targeting CDH7, CALR, APC and TGFβ1. Thus, the miRNA biomarkers investigated in the present study may represent novel therapeutic targets for the prediction and eventual improvement of the response to nCRT in patients with rectal cancer.

Tertiary lymphoid structures (TLSs) display phenotypic and functional characteristics of secondary lymphoid organs, and often develop in tissues affected by chronic inflammation, as well as in certain inflammation-associated cancers where they are prognostic of improved patient survival. However, the mechanisms that govern the development of tumour-associated TLSs remain ill-defined. Here, we observed tumour-associated TLSs in a preclinical mouse model (gp130

P16 is the product of cyclin-dependent kinase 2 (CDKN2A) gene and plays multi-pronged roles in the cancer progression. Breast cancer (BC) is the most commonly diagnosed cancer type among females. In the current study, the potential function of P16 in the growth and metastasis of BC was investigated. Firstly, the expression statuses of P16 in different cancer types were investigated using Oncomine database and validated with corresponding cancer cell lines. Afterwards, the expression of P16 was knocked down in BC cell line BT-549 and the effect on the cell proliferation, sensitivity to paclitaxel (TAX), apoptosis, migration, and invasion abilities was assessed using CCK-8, Edu, flow cytometry, scratch, and transwell assays, respectively. The influence of P16 inhibition and P16 overexpression on the activity of IL-6/JAK/STAT3 signaling was explored. Additionally, the effect of P16 inhibition on the tumor growth was verified with a BC xenograft mice model. The abnormal expression of P16 was detected in BC cell line BT-549 as well as colorectal cancer and osteosarcoma cell lines. The inhibition of P16 suppressed the cell proliferation, invasion, and migration abilities while induced the apoptosis and sensitivity to TAX in BT-549 cells. At molecular level, P16 knockdown inhibited the expression of IL6ST and Survivin, and the phosphorylation of JAK2 and STAT3. However, the induced expression of P16 in P16-knockdown BT-549 cells restored the activity of IL-6/JAK2/STAT3 pathway. The results of in vitro assays were confirmed with BC xenograft models: the inhibition of P16 decreased the tumor growth rate. Findings outlined in the current study demonstrated that the inhibition of P16 decreased the growth and metastasis potential of BC cells by inhibiting IL-6/JAK2/STAT3 signaling.

BACKGROUND: Large cell lung cancer (LCLC) patients have a poor prognosis because their tumors are highly malignant and show resistance to chemotherapy and radiotherapy. Plumbagin has anticancer activity toward several tumor types, but its effects on LCLC are unknown. This study investigated the effects of plumbagin on human L9981 and NL9980 large cell lung cancer cells and the mechanisms underlying its action.
METHODS: The effects of plumbagin on L9981 and NL9980 cells proliferative activity and invasion were analyzed using MTT and Boyden chamber assays, respectively. Exogenous IL-6 was used to detect the presence of the IL-6/STAT3 signaling pathway in LCLC cell lines. L9981 cells were harvested after plumbagin treatment at 9.0μmol/L (IC
RESULTS: After the introduction of exogenous IL-6, the mRNA expression of signaling genes and downstream genes was significantly increased in a concentration-dependent manner. Furthermore, plumbagin significantly inhibited the expression of the above mentioned genes in a dose-dependent and time-dependent manner. The mRNA expression levels of downstream genes were correlated with those of signaling genes.
CONCLUSION: Plumbagin was found to significantly inhibit the proliferation and invasion of L9981 and NL9980 cells, and may be an effective therapy for LCLC through targeting the IL-6/STAT3 signaling pathway.

Inflammasomes are key regulators of innate immunity in chronic inflammatory disorders and autoimmune diseases, but their role in inflammation-associated tumorigenesis remains ill-defined. Here we reveal a protumorigenic role in gastric cancer for the key inflammasome adaptor apoptosis-related speck-like protein containing a CARD (ASC) and its effector cytokine IL18. Genetic ablation of ASC in the

Annexin A7 (ANXA7) is a suppressor of tumorigenesis and metastasis in prostate cancer. Activated ANXA7 GTPase promotes prostate cancer cell apoptosis. However, the role and underlying mechanism of ANXA7 GTPase in prostate cancer metastasis have not been established. RKIP is a metastatic suppressor and downregulated in prostate cancer metastases. The binding of RKIP and its target proteins could inhibit the activation of its interactive partners. However, the effect of RKIP on ANXA7 GTPase activation is not clear. Here, we report that activation of ANXA7 GTPase by a small molecule SEC ((S)-ethyl 1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3- (4-methoxyphenyl)-1H-pyrazole-5-carboxylate) effectively inhibited prostate cancer metastasis. Mechanistically, activated ANXA7 promoted AMPK phosphorylation, leading to decreased mTORC1 activity, suppressed STAT3 nuclear translocation, and downregulation of pro-metastatic genes, including CCL2, APLN, and IL6ST. Conversely, RKIP interacted with ANXA7 and impaired activation of ANXA7 GTPase by SEC and its downstream signaling pathway. Notably, SEC treatment suppressed metastasis of prostate cancer cells in in vivo orthotopic analysis. Together, our findings provide a novel insight into how metastasis of prostate cancer with low RKIP expression is suppressed by SEC-induced activation of ANXA7 GTPase via the AMPK/mTORC1/STAT3 signaling pathway.

The purpose of the study was to test the efficacy of neoadjuvant palbociclib therapy and to evaluate its impact on cell cycle arrest and changes in EndoPredict (EP) scores before and after treatment. Postmenopausal women with histologically proven ER+ve, HER2-ve invasive breast cancer, 2 cm or greater, were enrolled in an open-label, single-arm study. Twenty eligible patients were given letrozole 2.5 mg per day together with palbociclib 125 mg per day for 3 out of 4 weeks in repeated cycles for 16 weeks (4 cycles) before surgery. The primary end points were clinical response rates (cRR) and preoperative endocrine prognostic index (PEPI). The secondary end points were pathologic response and gene expression testing with EP test on collected tumor samples. The following results were obtained. 17 patients showed a clinical response of 50% or more, including 8 complete responses and 9 partial responses. There was significant reduction in area (

BACKGROUND: Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families.
METHODS: The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student's T test was used for the statistical analysis of experimental replicates.
RESULTS: IL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation.
CONCLUSIONS: In conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ.

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.

PURPOSE: To compare the concordance in risk classification between the EndoPredict and the MammaPrint scores obtained for the same cancer samples on 40 estrogen-receptor positive/HER2-negative breast carcinomas.
METHODS: Formalin-fixed, paraffin-embedded invasive breast carcinoma tissues that were previously analyzed with MammaPrint as part of routine care of the patients, and were classified as high-risk (20 patients) and low-risk (20 patients), were selected to be analyzed by the EndoPredict assay, a second generation gene expression test that combines expression of 8 genes (EP score) with two clinicopathological variables (tumor size and nodal status, EPclin score).
RESULTS: The EP score classified 15 patients as low-risk and 25 patients as high-risk. EPclin re-classified 5 of the 25 EP high-risk patients into low-risk, resulting in a total of 20 high-risk and 20 low-risk tumors. EP score and MammaPrint score were significantly correlated (p = 0.008). Twelve of 20 samples classified as low-risk by MammaPrint were also low-risk by EP score (60%). 17 of 20 MammaPrint high-risk tumors were also high-risk by EP score. The overall concordance between EP score and MammaPrint was 72.5% (κ = 0.45, (95% CI, 0.182 to 0.718)). EPclin score also correlated with MammaPrint results (p = 0.004). Discrepancies between both tests occurred in 10 cases: 5 MammaPrint low-risk patients were classified as EPclin high-risk and 5 high-risk MammaPrint were classified as low-risk by EPclin and overall concordance of 75% (κ = 0.5, (95% CI, 0.232 to 0.768)).
CONCLUSIONS: This pilot study demonstrates a limited concordance between MammaPrint and EndoPredict. Differences in results could be explained by the inclusion of different gene sets in each platform, the use of different methodology, and the inclusion of clinicopathological parameters, such as tumor size and nodal status, in the EndoPredict test.

PBRM1 is a novel tumor suppressor gene that can inhibit cancer cell proliferation and predict the outcome of renal cell carcinoma (RCC), but its biological role needs further elucidation. We examined expression of the PBRM1 gene in RCC cell lines and the effect of PBRM1 on cell proliferation and cell cycle in RCC ACHN cells. Microarray processing and analysis was used to explore novel pathways involved in tumorigenesis related to PBRM1 knockdown. PBRM1 was expressed at high levels in RCC ACHN cells and lentivirus-mediated PBRM1 knockdown in these cells caused an increase in the proportion of cells in S phase of the cell cycle and promoted in vitro proliferation and migration. In vivo experiments showed that downregulation of PBRM1 promoted tumorigenesis in nude mice. In pathway gene chip analysis, the chemokine/chemokine receptor interaction pathway showed the greatest difference in gene expression upon PBRM1 knockdown. Protein levels of IL6ST and CCL2 were increased, whereas levels of interleukin (IL)-8, IL-6, and CXCL2 were decreased, in knockdown cells. Re-expression of IL-8 in PBRM1 knockdown ACHN cells could significantly decrease cell proliferation/migration and induced cell arrest in the G2/M phase. These findings indicate that PBRM1 alters cell cycle progression and inhibits proliferation and migration of ACHN cells through the chemokine/chemokine receptor pathway.

The hyperactivated Wnt/β-catenin signaling acts as a switch to induce epithelial to mesenchymal transition and promote colorectal cancer. However, due to its essential role in gut homeostasis, therapeutic targeting of this pathway has proven challenging. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways and key mucosal barrier components in regulating colon tumorigenesis and cancer progression remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue-specific role of claudin-3, a tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Loss of claudin-3 also predicted poor patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated role of the epigenetic regulation. Claudin-3-/- mice revealed dedifferentiated and leaky colonic epithelium, and developed invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, differentiated colon cancer in claudin-3-/- mice versus WT-mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune components. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation, which is further exacerbated by Stat-3-activation and help promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy.

Long noncoding RNAs can serve as oncogenes or tumor suppressors in human cancer; however, their biological functions and underlying mechanism in hepatocarcinogenesis are largely unknown. Here, we report a novel tumor suppressor long noncoding RNA on chromosome 8p12 (termed TSLNC8) that is frequently deleted and down-regulated in hepatocellular carcinoma (HCC) tissues. The loss of TSLNC8 is highly associated with the malignant features of HCC and serves as a prognostic indicator for HCC patients. TSLNC8 significantly suppresses the proliferation and metastasis of HCC cells in vitro and in vivo. TSLNC8 exerts its tumor suppressive activity by competitively interacting with transketolase and signal transducer and activator of transcription 3 (STAT3) and modulating the STAT3-Tyr705 and STAT3-Ser727 phosphorylation levels and STAT3 transcriptional activity, thus resulting in inactivation of the interleukin-6-STAT3 signaling pathway in HCC cells.
CONCLUSION: TSLNC8 is a promising prognostic predictor for patients with HCC, and the TSLNC8-transketolase-STAT3 axis is a potential therapeutic target for HCC treatment. (Hepatology 2018;67:171-187).

Interleukins-6 (IL-6)/GP130 signaling pathway represents a promising target for cancer therapy due to its critical role in survival and progression of multiple types of cancer. We have identified Bazedoxifene, a Food and Drug Administration (FDA)-approved drug used for the prevention of postmenopausal osteoporosis, with novel function as inhibitor of IL-6/GP130 interaction. In this study, we investigate the effect of Bazedoxifene in rhabdomyosarcoma and evaluate whether inhibiting IL-6/GP130 signaling is an effective therapeutic strategy for rhabdomyosarcoma. The inhibitory effect of Bazedoxifene was assessed in rhabdomyosarcoma cell lines in vitro and RH30 xenograft model was used to further examine the suppressive efficacy of Bazedoxifene on tumor growth in vivo. Rhabdomyosarcoma cells showed their sensitivity to GP130 inhibition using gene knockdown or neutralized antibody, suggesting IL-6/GP130 as therapeutic target in rhabdomyosarcoma cells. Bazedoxifene decreased the signal transducer and activator of transcription3 (STAT3) phosphorylation, blocked STAT3 DNA binding, and down-regulated the expression of STAT3 downstream genes. Bazedoxifene also induced cell apoptosis, reduced cell viability, and inhibited colony formation in rhabdomyosarcoma cells. The inhibition of colony formation, STAT3 phosphorylation, or cell viability following Bazedoxifene treatment was partially reversed by addition of excess IL-6 or overexpression of constitutive STAT3, respectively, supporting Bazedoxifene acted through IL-6/GP130 signaling. In addition, Bazedoxifene repressed cell invasion and angiogenesis in vitro. Furthermore, oral administration of Bazedoxifene significantly suppressed tumor growth and expression of STAT3 phosphorylation in nude mice bearing established human rhabdomyosarcoma xenograft. Taken together, these findings validate IL-6/GP130 signaling as therapeutic target in rhabdomyosarcoma and provide first evidence that Bazedoxifene may serve as a novel promising drug targeting IL-6/GP130 for treatment of rhabdomyosarcoma.

Mesenchymal stem cells (MSCs) interact with tumor cells and regulate tumorigenesis and metastasis. As one of the important components of the tumor microenvironment, MSC-secreted cytokines play a critical role in cancer development. However, whether and how bone marrow MSCs (BMSCs) and their secreted cytokines participate in hepatocellular carcinoma (HCC) progression, still remains largely unknown. In the present study, we first measured the concentration of interleukin-6 (IL-6) in BMSC conditioned medium (BMSC-CM). Next, we assessed the changes of invasion ability in response to treatment of BMSC-CM or recombinant IL-6 in two human HCC cell lines Bel-7404 and HepG2. Then we analyzed the level of key components of the IL-6 signal pathway, including IL-6 receptor and signal transducer (i.e. IL-6R and gp130), a transcription factor STAT3 (signal transducer and activator of transcription 3), as well as its target genes BCL2, CCND1, MCL1 and MMP2, in BMSC-CM or recombinant IL-6 treated Bel-7404 and HepG2 cells. Results showed that a considerable amount of IL-6 was secreted by BMSCs, and BMSC-CM markedly elevated Bel-7404 cell invasion rate and stimulated the signal transduction of IL-6/STAT3 pathway. Neutralizing the secreted IL-6 bioactivity by the anti-IL-6 antibody diminished the invasion-promoting effect and down-regulated IL-6/STAT3 pathway of BMSC-CM treated Bel-7404 cells. In conclusion, we found that BMSCs may activate the IL-6/STAT3 signaling pathway and promote cell invasion in Bel-7404 cells, suggesting that this protumor effect should be seriously considered before clinical application of MSC-mediated cancer therapy.

Surgery is the most effective treatment for breast cancer patients. However, some patients developed recurrence and distant metastasis after surgery. Adjuvant therapy is considered for high-risk patients depending on several prognostic markers, and lymphovascular invasion has become one of such prognostic markers that help physicians to identify the risk for distant metastasis and recurrence. However, the mechanism of lymphovascular invasion in breast cancer remains unknown. This study aims to unveil the genes and pathways that may involve in lymphovascular invasion in breast cancer. In total, 108 breast cancer samples were collected during surgery and microarray analysis was performed. Significance analysis of the microarrays and limma package for R were used to examine differentially expressed genes between lymphovascular invasion-positive and lymphovascular invasion-negative cases. Network and pathway analyses were mapped using the Ingenuity Pathway Analysis and the Database for Annotation, Visualization and Integrated Discovery. In total, 86 differentially expressed genes, including 37 downregulated genes and 49 upregulated genes were identified in lymphovascular invasion-positive patients. Among these genes, TNFSF11, IL6ST, and EPAS1 play important roles in cytokine-receptor interaction, which is the most enriched pathway related to lymphovascular invasion. Moreover, the results also suggested that an imbalance between extracellular matrix components and tumor micro-environment could induce lymphovascular invasion. Our study evaluated the underlying mechanisms of lymphovascular invasion, which may further help to assess the risk of breast cancer progression and identify potential targets of adjuvant treatment.

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins.

Toll-like receptors (TLRs) are key regulators of innate immune responses, and their dysregulation is observed in numerous inflammation-associated malignancies, including gastric cancer (GC). However, the identity of specific TLRs and their molecular targets which promote the pathogenesis of human GC is ill-defined. Here, we sought to determine the clinical utility of TLR2 in human GC. TLR2 mRNA and protein expression levels were elevated in >50% of GC patient tumors across multiple ethnicities. TLR2 was also widely expressed among human GC cell lines, and DNA microarray-based expression profiling demonstrated that the TLR2-induced growth responsiveness of human GC cells corresponded with the up-regulation of six anti-apoptotic (BCL2A1, BCL2, BIRC3, CFLAR, IER3, TNFAIP3) and down-regulation of two tumor suppressor (PDCD4, TP53INP1) genes. The TLR2-mediated regulation of these anti-apoptotic and tumor suppressor genes was also supported by their increased and reduced expression, respectively, in two independent genetic GC mouse models (gp130

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

BACKGROUND: Inflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC.
METHODS: We characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student's t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey's multiple comparison tests.
RESULTS: Our data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44
CONCLUSIONS: Syndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.

Loss of tumor suppressor adenomatous polyposis coli (APC) activates β-catenin to initiate colorectal tumorigenesis. However, β-catenin (

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 & AIMS: Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in β-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications.
METHODS: We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation.
RESULTS: Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with β-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively.
CONCLUSIONS: Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA, disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients with HCA.

Lung cancer is the leading cause of cancer death worldwide, and is frequently associated with the devastating paraneoplastic syndrome of cachexia. The potent immunomodulatory cytokine interleukin (IL)-6 has been linked with the development of lung cancer as well as cachexia; however, the mechanisms by which IL-6 promotes muscle wasting in lung cancer cachexia are ill-defined. In this study, we report that the gp130