Induce Tumor Progression Research Articles

TNF-α enhances TGF-β-induced endothelial-to-mesenchymal transition via TGF-β signal augmentation.

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.

Anticancer activity of a 1,4-dihydropyridine in DMBA-induced mouse skin tumor model.

Antitumor potential of a 1,4-dihydropyridine derivative (DHP-8) has been successfully studied previously in a number of cancer cell lines including the human melanoma cells, A375. In order to validate its anticancer activity, DMBA induced tumor in Swiss Albino mice was considered for this study. DMBA causes skin carcinoma in murine systems and is an important in vivo model for evaluating the efficacy of any new chemical entity against skin cancer. Topical administration of DHP-8 at the dose rate of 33.3 and 50.0 mg/kg body weight showed a significant reduction in tumor parameters. It also prevented the progression and differentiation of squamous cell carcinoma, as evidenced from histopathological studies. Immunohistochemical analysis for the expression of Ki67 indicated that it also reduced cancer cell proliferation. Additionally, it induced apoptosis in the tumor cells by activation of Caspase3. Our results indicated that DHP-8 efficiently attenuated DMBA induced tumor progression and it could be a potent therapeutic agent for skin cancer treatment.

Incomplete ablation of colon cancer cells may induce activation of dormant cells: Evidence from bioinformatics analysis.

It is not yet verified whether incomplete radiofrequency ablation (iRFA) induces tumor progression and hypoxia related to tumor dormancy. This study showed the relationship between iRFA and tumor dormancy. To identify the candidate genes in the control and iRFA-treated colon cancer cells, microarray datasets GSE138224 were downloaded from Gene Expression Omnibus database. Using NetworkAnalyst, the differentially expressed genes (DEGs) were identified, function enrichment analyses were performed, and the protein-protein interaction (PPI) network and key PPI network were constructed. A total of 656 DEGs were identified, comprising 637 downregulated and 19 upregulated genes. The enriched functions and pathways of the upregulated DEGs include an immune effector process, regulation of tyrosine phosphorylation of signal transducer and activator of transcription (STAT) protein, tyrosine phosphorylation of STAT protein, JAK-STAT cascade, and regulating JAK-STAT cascade, and CCL5 gene participated in regulating the JAK-STAT signaling pathway. The downregulated DEGs were mainly enriched in extracellular matrix-receptor interaction, PI3K-Akt signaling, Wnt signaling, transforming growth factor-beta signaling, and mitogen-activated protein kinase signaling pathways. There are three key PPI networks of DEGs (degree ≥10 and hub genes >3). The dormancy-related genes Bmp4 and Ccl5 were regarded as hub genes in the PPI network with Bmp4 as a downregulated gene and CCL5 as an upregulated gene. The identified DEGs and function enrichment analyses in this study aid the understanding of molecular mechanisms underlying the relationship between iRFA and tumor dormancy.

MYC Overexpressing Multiple Myeloma Are Dependent on GLS1

Introduction. MYC alterations trigger transition from monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to multiple myeloma (MM). They also represent secondary genomic events inducing tumor progression. MYC localization to the nucleus and the short life of the protein are key factors that limit its direct targeting. To overcome these issues, we sought to determine the top genomic dependencies in MYC overexpressing MM by analyzing large-scale knockdown screening, followed by functional validations. Methods. We performed in silico analyses from the Dependency Map (Achilles 2.4.3) together with CCLE (Affymetrix U133+2 expression array), CLUE (Connectivity Map) and MM patient datasets (Chng et al. 2007, Gutiérrez et al. 2010, MMRF RG Dataset), to look for gene dependencies and differentially expressed pathways in MYC OE cancer cell lines and MM patient samples. We generated an isogenic model of MYC OE in U266 MM cell line by using EF1A-C-MYC lentiviral vector, and performed RNA sequencing, a quantitative proteomic analysis by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. To further investigate dependency on glutamine metabolism in MYC OE cell lines, we treated them with GLS1 inhibitor CB-839 and siRNA targeting GLS1 in several cell lines with various MYC expressions and in our isogenic model. Results. By analyzing correlations between MYC expression and gene ATARiS scores corresponding to the effect of over 9000 knockdowns in 236 cell lines, we identified specific vulnerabilities of MYC overexpressing cells for the genes involved in glutamine metabolism and cell cycle pathways. Top dependencies were observed with MYC binding protein MAX (r = -0.51, p < .001), representing an internal control as it is a co-activator of MYC, followed by GLS1 (r = -0.48, p < .001) and SLC1A1 (r = -0.42, p < .001), both involved in glutamine metabolism, together with E2F6 (r = -0.41, p < .001), involved in cell cycle. To further validate dependencies obtained from Achilles data, we generated an isogenic model of MYC OE in U266 (a low c-myc expressing MM cell line). GSEA analysis of RNA seq data showed strong enrichments of translation and cell cycle pathways, with similar results observed in CCLE and MM patient data. Quantitative proteomics analysis of U266 isogenic model showed overexpression of genes involved in glutamine transport (SLC1A5; FC = 1.28, p < .05), glucose metabolism (HK2; FC = 3.68, p < .001) and cell cycle progression (CDK6; FC = 2.85, p < .001). To explore the therapeutic potential of these dependencies, we performed a primary screen of 1902 small-molecules and identified 47 compounds with potent activity on U266/MYC model. Validation screen of these hits identified three leading compounds to which U266/MYC cells showed highest sensitivity at 10 µM concentration - Torin-2 (U266/C 40.28 ± 6.74% vs. U266/MYC 16.05 ± 3.21%), LY2835219 (U266/C 52.70 ± 9.63% vs. U266/MYC 5.52 ± 0.89%) and AT7519 (U266/C 43.03 ± 4.02% vs. U266/MYC 30.13 ± 4.90%), targeting proteins involved in translation and cell cycle pathways. For the functional validation of GLS1 dependency in MYC overexpressing cells, MYC OE cell lines were treated with GLS1 inhibitors CB-839 and 968. MYC high MM cell lines showed higher sensitivity to CB-839 inhibitor compared to MYC low cell lines at 1 µM concentration, after 48 (KMS-12-BM 14.19 ± 0.07%, KMS-18 31.56 ± 2.84%, MM.1S 23.21 ± 1.21% vs. NCI-H1650 46.49 ± 3.48%, U266 52.72 ± 4.99%, LOUCY 37.14 ± 1.14%, OVCAR-3 64.14 ± 5.19%) and 72 h (KMS-18 19.69 ± 3.15%, MM.1S 15.09 ± 1.28% vs. NCI-H1650 34.82 ± 0.95%, U266 61.73 ± 1.70%, LOUCY 46.27 ± 6.27%, OVCAR-3 65.34 ± 1.23%). This suggests that GLS1 dependency in MYC OE cells offers a therapeutic window for the use of GLS1 inhibitors in MM. Conclusion. By using a combination of different datasets and models, we characterized the main dependencies in MYC overexpressing MM. Glutamine metabolism and cell cycle emerged as strong dependencies by using therapeutic inhibitors. Altogether, our results demonstrate that MYC OE MM cells are dependent on glutamine metabolism and cell cycle, and these findings can potentially lead to development of new therapeutic approaches in MM patients. Disclosures Leleu: Oncopeptide: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Carsgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Karyopharm: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Merck: Honoraria. Facon:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Manier:Amgen: Research Funding; Celgene: Research Funding; Janssen: Research Funding.

CSIG-19. CYTOMEGALOVIRUS INFECTION LEADS TO NF-kB DEPENDENT UPREGULATION OF c-MET AND MGMT IN GLIOBLASTOMA AND RESISTANCE TO TEMOZOLOMIDE IN VIVO

Abstract INTRODUCTION Cytomegalovirus (CMV), a member of the betaherpesvirinae subfamily widespread in human populations has been reported to be detectable in glioblastoma (GBM). We have recently shown that CMV latent infection promotes GBM growth in a mouse model. The mechanisms involved are not well-defined and treatment responses have not been evaluated. METHODS To investigate the effects of CMV on GBM cells we performed an RNAseq study. Differentially expressed genes were identified and validated in vitro and in vivo. RESULTS RNAseq analysis showed multiple alterations induced by CMV infection of GBM cells including significant upregulation of c-MET (8.2 log2 fold change) and MGMT (3.9 log2 fold change) transcripts after CMV infection. c-MET is a tyrosine kinase receptor known to promote GBM growth, and MGMT expression leads to resistance to the alkylating agent temozolomide (TMZ). These findings were validated in vitro in both mouse and human GBM cells, and shown to be dependent on CMV-induced upregulation of NF-kB by siRNA knockdown of the essential RelA NF-kB subunit. The c-MET co-receptor CD44 was also upregulated suggesting a role for the c-MET signalling axis in CMV induced tumor progression. CMV-infected tumor cells were resistant to TMZ treatment as expected due to MGMT upregulation. Treatment of our murine GBM model with TMZ led to a significant increase in survival in control mock-infected mice (median survival 38 days control, 48 days TMZ), whereas the CMV infected mice did not respond (median survival 35 days control, 35 days TMZ; p value between TMZ treated groups = 10–4). CONCLUSIONS Our results define novel and relevant mechanisms by which CMV may influence both GBM growth and resistance to treatment. Further therapeutic and mechanistic studies are underway to investigate these pathways in the context of CMV in GBM. These data support further study of CMV targeting in GBM.

Melanoma-Derived Exosomes Induce PD-1 Overexpression and Tumor Progression via Mesenchymal Stem Cell Oncogenic Reprogramming.

Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive. However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations. Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSCPD-1+) from naïve mesenchymal stem cells (MSCs). Exosomes and mMSCPD-1+ cells induce tumor progression and expression of oncogenic factors in vivo. Finally, we revealed a characteristic, tumorigenic signaling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, MTOR) and their upstream exosomal regulating proteins and miRNAs. Our study highlights the complexity of exosomal communication during tumor progression and contributes to the detailed understanding of metastatic processes.

Abstract 3555: miR-374a-5p induces tumor progression in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) has higher aggressiveness and poorer outcomes compared with other subtypes of breast cancer. However, the genomic and molecular aberrations of TNBC are largely unknown, with cytotoxic chemotherapy remaining the major treatment for TNBC patients. In this study, miR-374a-5p was discovered as a novel TNBC-specific miRNA and its functions and the molecular mechanisms involved were investigated. Combined gene expression profiling of miRNA-microarray and human transcriptome dataset analysis revealed that miR-374a-5p is specifically upregulated in TNBC patients. Functional studies using in vitro and in vivo TNBC models, as well as survival analysis of TNBC patients, indicated that upregulated miR-374a-5p promotes tumor progression in TNBC. miR-374a-5p was also found to directly target arrestin beta 1 (ARRB1) that is specifically downregulated in TNBC patients in several human genomic datasets. Overexpressed ARRB1 reduced TNBC cell growth and migration, and the ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumor-suppressive function of ARRB1 in breast cancer. Interestingly, increased ARRB1 activates AMPK in TNBC cells, which is associated with the expression of miR-374a-5p. Taken together, the findings suggest that miR-374a-5p is a novel prognostic marker and therapeutic target in the treatment of TNBC. Citation Format: Yesol Kim, Dasom Son, Do Yeon Kim, Jong Hoon Park. miR-374a-5p induces tumor progression in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3555.

SAT-569 Nuclear TGF-β1 Expression Identifies More Aggressive Thyroid Tumors

TGF-β1 is a member of the Transforming Growth Factor β (TGF-β) superfamily of cytokines that is involved in proliferation, migration, differentiation and apoptosis of different cell types. This cytokine can act as a tumor suppressor in early stages of cancer, controlling cellular proliferation and growth; however, as cancer progresses, tumor cells lose their sensitivity to the inhibitory effects of TGF-β1, and, together with increased angiogenesis, induces tumor progression. In order to evaluate a possible clinical utility of TGF-β1 staining, we made a Tissue MicroArray (TMA) including 136 benign (113 female, 18 male, 49.1±14.6 years old), 154 malignant (118 female, 28 male, 42.6±17.0 years old) and 11 normal thyroid tissues. The patients were followed-up for 75.3±65 months. There were 122 goiters, 14 follicular adenomas (FA), 138 differentiated thyroid carcinomas (DTC) [73 classic papillary thyroid carcinomas (CPTC), 42 follicular variant of PTC (FVPTC), 23 follicular thyroid carcinomas (FTC)] and 16 poorly differentiated thyroid carcinomas (PDTC). The analysis were performed by Allred Score with two pathologists independent evaluation. Positive/negative score for cytoplasmic TGF-β1 expression was not different between benign and malignant tissues (p=0.4334). For nuclear TGF-β1 expression, the percentage positive/negative score was 90/10% of benign tissues and 71/29% of malignant (p<0.0001). In fact, nuclear positivity decreased according to the tumor dedifferentiation. We found that 91% of normal tissues presented nuclear positivity, decreasing to 90% of the goiters, 86% of the FA, 74% of the DTC and 44% of the PDTC. Nuclear TGF-β1 positive/negative score was able to distinguish PDTC from DTC (p=0.0188), CPTC (p=0.0384), FTC (p=0.0172), AF (p=0.0259) and goiter (<0.0001). Although there was a net correlation between nuclear TGF-β1 positivity with aggressive thyroid tumors, we were unable to demonstrate any relationship with clinical or pathological features of the tumors. In conclusion, our data suggest that evaluation of nuclear TGF-β1 expression may help identify more aggressive thyroid tumors. Sources of Research Support: Foundation for Research of the State of São Paulo (FAPESP) scholarship [grant number 2015/19117-0] to KCP.

TRIM44 activates the AKT/mTOR signal pathway to induce melanoma progression by stabilizing TLR4

BackgroundThere is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development. However, the underlying mechanism of this deubiquitinating enzyme remains unclear.MethodsLarge clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis. Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression. Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44.ResultsWe found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues. TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002). Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients. Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo. Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis. Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression. Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression. Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44.ConclusionsTRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway. TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.

Integrin α11 in pancreatic stellate cells regulates tumor stroma interaction in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest tumor due to its highly abundant tumor stroma. Pancreatic stellate cells (PSCs) are considered precursor cells of cancer-associated fibroblasts (CAFs), which induce tumor progression, invasion, and metastasis. In this study, we investigated the role of integrin subunit α (ITGA) 11, the receptor for collagen type I, in tumor stroma interaction. Clinical sample analysis showed that ITGA11 was overexpressed by CAFs in PDAC stroma, as shown with colocalization immunostaining with α-smooth muscle actin. In contrast, there was no expression in healthy pancreas. Public transcriptomic data confirmed a reduced expression of ITGA11 in healthy pancreas and adjacent nontumoral tissues compared with human tumor tissues. Primary human PSCs (hPSCs) activated with either TGF-β or pancreatic cancer cell (PANC-1)-conditioned medium (CM) resulted in the significant up-regulation of ITGA11 and various CAF markers. Furthermore, short hairpin RNA (shRNA)-mediated stable ITGA11 knockdown (shITGA11) in hPSCs significantly inhibited TGF-β- and PANC-1 CM-mediated activation at both gene and protein levels of extracellular matrix, cytokines, and adhesion molecules. Additionally, shITGA11 hPSCs had a reduced migration and contractility compared with shRNA control (shCTR) PSCs. Furthermore, we investigated the effect of ITGA11 on the paracrine effects of hPSCs. Interestingly, the CM from shITGA11 hPSCs, activated with either TGF-β or PANC-1 CM, caused tumor cells to migrate and invade lesser compared with their counterpart, activated shCTR PSCs. In summary, this study presents ITGA11 as an interesting stromal therapeutic target that plays a crucial role in the regulation of the differentiation of PSCs into CAFs and paracrine effects.-Schnittert, J., Bansal, R., Mardhian, D. F., van Baarlen, J., Östman, A., Prakash, J. Integrin α11 in pancreatic stellate cells regulates tumor stroma interaction in pancreatic cancer.

Targeted regulation by ROCK2 on bladder carcinoma via Wnt signaling under hypoxia.

Bladder cancer is frequently occurred in urinary system and has complicated pathogenesis factors including both genetics and environmental factors that have not been fully illustrated. Hypoxia can further induce tumor progression. ROCK2 has abnormal expression in various tumors but its expression or functional role in bladder cancer have not been illustrated. In vitro cultured bladder cancer cell line T24 was randomly assigned into control group, hypoxia group (prepared under hypoxic culture), and ROCK2 siRNA group (transfected with ROCK2 siRNA after hypoxia treatment). Real-time PCR and Western bot measured ROCK2 expression. MTT assay tested cell proliferation, and cell migration was quantified. Cell apoptosis was measured by caspase3 activity assay kit and Transwell chamber measured cell migration. Western blot quantified expressional change of HIF-1α and E-cadherin, and Wnt signal pathway proteins including Wnt4, and β-catenin. ROCK2 is up-regulated in bladder cancer T24 cells under hypoxia, and can facilitate cell proliferation, migration and invasion, inhibited Caspase3 activity, enhanced HIF-1α expression, decreased E-cadherin expression, and up-regulated Wnt4 and β-catenin (p< 0.05 comparing to hypoxia group). Under hypoxia conditions, ROCK2 can facilitate apoptosis of bladder cancer cells via modulating Wnt signal pathway, inhibit cell proliferation, migration, invasion or formation of epithelial mesenchymal transition (EMT).

The Role of Androgen Receptor Signaling in Ovarian Cancer.

Emerging evidence has suggested that androgen receptor signaling plays an important role in ovarian cancer outgrowth. Specifically, androgen receptor activation appears to be associated with increased risks of developing ovarian cancer and inducing tumor progression. However, conflicting findings have also been reported. This review summarizes and discusses the available data indicating the involvement of androgens as well as androgen receptor and related signals in ovarian carcinogenesis and cancer growth. Although the underlying molecular mechanisms for androgen receptor functions in ovarian cancer remain far from being fully understood, current observations may offer effective chemopreventive and therapeutic approaches, via modulation of androgen receptor activity, against ovarian cancer. Indeed, several clinical trials have been conducted to determine the efficacy of androgen deprivation therapy in patients with ovarian cancer.

F-box/WD-40 repeat-containing protein 7: A potential target in the progression and treatment of gastrointestinal malignancy.

Cancer is a principal cause of human morbidity and mortality, with gastrointestinal malignancies, in particular, resulting in a marked number of tumor-associated mortalities. The progression of gastrointestinal malignancy is regulated by a variety of aberrantly expressed proteins, a number of which facilitate tumor progression, whereas, others function as tumor suppressors. The detection of such proteins not only contributes to the early diagnosis of cancer, they may additionally serve as potential therapeutic targets. In normal tissues, numerous proteins encoded by proto-oncoproteins are degraded by ubiquitylation enzymes, consisting of F-box/WD-40 repeat-containing protein 7 (Fbw7) and other proteins, thus avoiding tumorigenesis and maintaining homeostasis. In tumor tissues, the downregulation of Fbw7, caused by various factors, leads to disorders in ubiquitinase synthesis, which may induce tumor progression and chemoresistance, particularly in gastrointestinal malignancy. Therefore, an in-depth study of the regulatory mechanisms involved in disorders of Fbw7 expression and the role of Fbw7 in chemoresistance of gastrointestinal tumors may suggest improvements for early diagnostic screening and targeted therapy.

Experimental and clinicopathological analysis of HOXB9 in gastric cancer.

The association between homeobox (HOX)B9 expression and tumor malignancy was identified recently. It was reported that HOXB9 induced tumor angiogenesis, and associated with poor prognosis in patients with breast and colon cancer. On the other hand, regional lymph nodes are the most common site of tumor spread, and lymph node metastasis is a major prognostic factor in gastric cancer. It was hypothesized that HOXB9 promotes tumor lymphangiogenesis and induces tumor progression, invasion and metastasis in gastric cancer. The aim of the present study was to evaluate the correlation between HOXB9 expression, prognosis and clinicopathologic factors in patients with gastric cancer, and to assess the contribution of HOXB9 expression to tumor cell lymphangiogenesis in vitro. HOXB9 expression was evaluated by immunohistochemistry in resected tumor tissues from 58 patients with gastric cancer, and the association between prognosis and clinicopathologic factors was determined. HOXB9 gene was overexpressed in human gastric cancer TMK-1 cells and the effect of HOXB9 overexpression on the expression of vascular endothelial growth factor (VEGF)-C, VEGF-D and VEGF receptor (R)-3 was determined. It was demonstrated that the depth of tumor invasion, the number of node metastases, lymphatic invasion and vascular invasion were significantly associated with HOXB9 expression. Overall survival was decreased in patients with HOXB9 expression. The mRNA expression of VEGF-D but not of VEGF-C and VEGFR-3 was increased in HOXB9-overexpressing TMK-1 cells compared with control cells. In conclusion, HOXB9 expression was positively correlated with gastric cancer progression and lymphangiogenesis marker expression. HOXB9 may be associated with lymphogenic metastasis.

Low expression of pentraxin 3 and nuclear factor-like 2 implying a relatively longer overall survival time in gliomas.

Pentraxin 3 (PTX3) and nuclear factor-like 2 (Nrf2) are known to induce tumor progression in certain malignancies but act as tumor suppressors in other human neoplasms. In this study, we not only tested the association between PTX3 expression and the World Health Organization (WHO) tumor grading system but also evaluated overall patient survival under variable expression of PTX3 and Nrf2 in primary brain tumors (PBTs). Immunohistochemistry (IHC) was performed for PTX3 and Nrf2 in 10 nonneoplastic brain tissues and 197 PBTs. IHC scores were calculated as the degree of cytoplasmic and nuclear PTX3 and Nrf2 staining intensity multiplied by the percentage of positively stained tissue area. The correlation between PTX3 and Nrf2 IHC scores and tumor grades as well as overall survival time was analyzed by Pearson product-moment correlation and Kaplan-Meier estimate. According to our results, PTX3 IHC scores showed a positive correlation with the WHO grades of gliomas and meningiomas. In addition, we also observed that higher PTX3 expression was associated with poor prognosis in gliomas but not in meningiomas. The concordance between PTX3 and Nrf2 immunohistochemistry (IHC) scores was analyzed using linear regression analysis. When compared to groups with high IHC scores for either one or both biomarkers, gliomas with low expression of both PTX3 and Nrf2 showed significantly better prognosis. In conclusion, we demonstrated that high PTX3 expression implied aggressive tumor behavior and shorter survival time in glioma patients. In addition, our results also showed that gliomas with low PTX3 and Nrf2 immunohistochemical expression could imply a longer overall survival time. Therefore, the combination of lower PTX3 and Nrf2 immunohistochemical expression may be important in offering a better prognosis in gliomas, although the detailed mechanism is yet to be elucidated.

FOXD4 induces tumor progression in colorectal cancer by regulation of the SNAI3/CDH1 axis

ABSTRACTColorectal cancer (CRC) is ranked third as the most common malignancy, and it develops into metastasis at a high rate. Importantly, distant metastasis is considered to be a key factor for colorectal therapy. In the present study, we identified FOXD4, a transcription factor belonging to the forkhead/winged helix-box (FOX) family, as a novel biomarker for diagnosis and treatment of patients with CRC. We revealed that FOXD4 was up-regulated in CRC tissues and increased the metastatic ability of CRC cells. Additionally, FOXD4 affected the metastasis of CRC by inducing the epithelial-mesenchymal transition (EMT) process. Furthermore, FOXD4 could directly bind the SNAI3 promoter during EMT in CRC and then facilitate CRC metastasis. In summary, the present research strongly suggests that FOXD4 is a valuable marker for CRC, and that targeting FOXD4 may be a novel strategy for enhancing the treatment outcomes of CRC therapy

Receptor activator of NF-κB (RANK)-mediated induction of metastatic spread and association with poor prognosis in renal cell carcinoma

BackgroundInhibition of the receptor activator of NF-κB ligand (RANKL) has become a standard of care supportive treatment to prevent skeletal related events in cancer patients. Moreover, RANKL inhibition has been implicated with better survival outcome in lung cancer, while RANKL expression induces tumor progression and metastatic spread in vivo in breast cancer. Whether RANK/RANKL may have an impact on the pathogenesis of clear cell renal cell carcinoma (ccRCC) is currently unknown. Patients and MethodsA retrospective tissue micro array (TMA)-study was carried out determining the expression of RANK/RANKL in primary tumors of 306 ccRCC patients. Additionally, 24 ccRCC cell lines were employed for in vitro analyses of the RANK/RANKL axis including cell proliferation, migration and anchorage independent growth. ResultsRANK (+) vs. RANK (−) tumors had both worse cancer specific survival (CSS) (6.3 vs. 1.3 years; p < 0.001) and recurrence free survival (RFS) (9.9 vs. 5.8 years; p < 0.001). RANK (+) (HR 2.21; p < 0.001) was an independent prognostic factor for CSS and RFS (HR 4.98; p < 0.001). RANKL treatment resulted in increased proliferation, soft agar growth, and colony formation of RANK (+) RCC cell lines, which could be reversed by treatment with an NF-κB inhibitor and with a combination of osteoprotegrin and RANKL in vitro. ConclusionsRANK is expressed in ccRCC tissue, correlates with clinicopathological features, survival outcome, and when stimulated with RANKL can induce ccRCC progression in vitro. Consequently, RANKL inhibition combined with standard of care treatment may be a promising approach to improve ccRCC patient's survival.

Abstract 2538: The interaction of IGFBP3 with HIF2α in the invasion regulation of ovarian epithelial cell cancer

Abstract Ovarian cancer is the most lethal gynecological cancer among women worldwide. The majority of malignant ovarian tumors are epithelial ovarian cancer and metastasis occurred in most patients at the time of diagnosis with worse outcome. New target therapy such as anti-angiogenesis therapy was proved to be effective to prolong progression free survival in epithelial ovarian cancer (EOC) patients. In our previous study, we have established an ovarian endometrioid carcinoma cell line OVTW59. By cDNA microarray analysis and further functional analysis, we have identified Insulin-like Growth Factor Binding Protein-3 (IGFBP-3), Calreticulin (CRT) and Thrombospondin-1(THBS1) correlated with tumor invasion in OVTW59. IGFBP3 and CRT was further found related with poor patient survival outcome. In this study, we focused on the mechanism of IGFBP3 in cancer invasion and metastasis. First we focused on the THBS1 related angiogenesis mechanism which was induced by hypoxia. Through this study, we identified HIF-2α, but not HIF-1α, playing as a significant role in EOC. By q-RT-PCR, Western blotting and immunohistochemical staining, HIF-2α showed opposite expression with IGFBP3, and high IGFBP3 was correlated with high THBS1 expression and low CRT expression. Using other cell lines, the IGFBP3/THBS1/HIF-2α/CRT expression patterns were similar in 293T, H1299 and A549. Furthermore, by Luciferase promoter assay, we verified that IGFBP3 could induce THBS1, inhibit HIF-2α expression, but does not regulate the expression of CRT. IGFBP3 is known as an invasion suppressor gene, THBS1 is an endogeneous inhibitor of angiogenesis, HIF-2α is a regulator of the hypoxia response and angiogenesis inducer, and CRT as a cell adhesion and invasion related protein. Overall, our study shows that IGFBP3 could induce tumor progression and metastasis through direct regulation of THBS1, and HIF-2α and indirect regulation of CRT in EOC. Citation Format: Ho-Jun Shih. The interaction of IGFBP3 with HIF2α in the invasion regulation of ovarian epithelial cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2538.

Abstract LB-079: The interaction of MSI1 and AGO2 promotes glioblastoma multiforme progression under environmental stress

Abstract Glioblastoma multiforme (GBM) is the high-grade astrocytoma; it is the most common and aggressive malignant brain tumor. GBM is largely resistant to therapy and experiences a corresponding poor prognosis. Therefore, GBM progression and recurrence are the main therapeutic challenges frequently faced by patients with refractory tumors. Nevertheless, the underlined molecular mechanism remains unknown. The RNA-binding protein musashi1 (MSI1) is expressed in stem cell compartments and in the aggressive tumor, only little is known about its function and mechanism in fact decision of target mRNA and post-transcription control. In this study, we describe a mechanism by which stress induces tumor progression via elevated cytoplasmic translocation of MSI1 in malignant neoplasms. MSI1 forms ribonucleoprotein (RNP) complexes with its downstream mRNAs at the 3'-untranslated region (3'UTR) or coding domain sequence (CDS) in the nucleus. Upon stress conditions, the MSI1/mRNA complex translocates to the cytosol where Argonaute2 (AGO2) is recruited and directly interact with MSI1. The MSI1/AGO2 complex modulates mRNA stability depending on their binding locations on the mRNA targets. Blocking of MSI1 shuttling by either a nuclear export inhibitor (Leptomycin B, LMB) or NES/NLS mutant attenuates GBM tumor growth in the mouse model. Importantly, the MSI1-AGO2 complex enhances GBM cells proliferation and survival under stress stimulation. MSI1 directly binds to AGO2 in the cytosol through its C-terminus. Orthotopic delivery of MSI1-C-terminus decoy peptides, which disrupt the MSI1/AGO2 interaction, effectively reduces stress-induced tumor progression. These results provide new insights into the function of MSI1 and suggest a novel peptide-based therapeutic strategy against refractory malignancies. Citation Format: Hsiao-yun Chen, Shih-Hwa Chiou. The interaction of MSI1 and AGO2 promotes glioblastoma multiforme progression under environmental stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-079.

Role of Kupffer cells in the progression of CRC liver metastases after the first stage of ALPPS

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been suggested as a potential therapy for extensive bilobar liver tumors, although in some circumstances this technique may induce tumor progression, a fact still not well studied. Our aim was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular CC531 cell line inoculation. ALPPS induced: tumor progression on deportalized lobe and metastases; expression of hepatic vasculogenic factors (HIF1-α and VEGF); and a dramatic increase of Kupffer cells (KCs) and tumor-associated macrophages (TAMs). Interestingly, KCs expressed COX-2 (M1 polarization), while TAMs expressed mainly arginase-1 (M2 polarization). ALPPS also induced a decrease of tumor-infiltrating lymphocytes and an increase of intrahepatic T lymphocytes. Thus, ALPPS technique seems to induce a hypoxic environment, which enhances hepatic HIF1-α and VEGF expression and may promote KCs and TAMs polarization. Consequently, the regenerative stimulus seems to be driven by a pro-inflammatory and hypoxic environment, in which M1 intrahepatic macrophages expressing COX-2 and T-Lymphocytes play a key role, facts which may be related with the tumor progression observed.