Regulation Of Tumor Progression Research Articles

MicroRNA-761 is downregulated in colorectal cancer and regulates tumor progression by targeting Rab3D.

The purpose of the present study was to investigate the biological role of microRNA-761 (miR-761) in colorectal cancer (CRC) and the underlying mechanisms by which miR-761 regulates CRC cell proliferation and migration. Quantitative polymerase chain reaction was performed to measure miR-761 expression in CRC tumor tissues and cell lines. It was demonstrated that miR-761 expression was dramatically reduced in CRC tumor tissues and cell lines compared with in normal tissues and cell lines. Overexpression of miR-761 significantly decreased CRC cell growth and migration. Using bioinformatics analysis and luciferase reporter assays, Rab3D was identified as a novel target of miR-761. In addition, it was demonstrated that Rab3D expression was negatively correlated with miR-761. Furthermore, overexpression of Rab3D could reverse the inhibitory effects of miR-761 on cell proliferation and migration. Collectively, the present study demonstrated that miR-761 overexpression could inhibit the proliferation and migration of CRC cell lines, partly at least, via directly targeting Rab3D.

Deletion 6q Drives T-cell Leukemia Progression by Ribosome Modulation.

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.

CircVRK1 regulates tumor progression and radioresistance in esophageal squamous cell carcinoma by regulating miR-624-3p/PTEN/PI3K/AKT signaling pathway

As a novel class of noncoding RNAs (ncRNAs), circular RNAs (circRNAs) have been verified to be potential biomarkers and therapeutic targets for human malignant tumors. However, the thorough understanding of circRNAs in the progression of esophageal squamous cell carcinoma (ESCC) still needs to be improved. This study focused on exploring the function and mechanism of circVRK1 in ESCC. At first, we examined the expression level of circVRK1 in ESCC tissues and cell lines with qRT-PCR. We found that circVRK1 was downregulated in ESCC tissues and cell lines. Kaplan-Meier method was used to analyze the correlation between circVRK1 expression and the overall survival of ESCC patients. Functionally, overexpression of circVRK1 suppressed the cell proliferation, migration and epithelial-mesenchymal transition (EMT) and reversed the radioresistance. Therefore, we identified the tumor suppressive role of circVRK1 in ESCC progression. Mechanistically, circVRK1 positively regulated PTEN by acting as a molecular sponge of miR-624-3p. Moreover, circVRK1 decreased the activity of PI3K/AKT signaling pathway by upregulating PTEN. Rescue assays were carried out to confirm the function of circVRK1-miR-624-3p-PTEN axis in ESCC progression. Our findings showed that circVRK1 suppressed ESCC progression by regulating miR-624-3p/PTEN axis and PI3K/AKT signaling pathway, suggesting the potential therapeutic value of circVRK1 for ESCC.

Tissue Factor Pathway Inhibitor Enhances Transendothelial Migration of Chronic Lymphocytic Leukemia Cells through Binding to Glypican-3

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Cell trafficking and homing of CLL cells play a critical role in organ infiltration and contribute to the clinical course of CLL. Interaction between CLL cells and endothelial cells affects gene expression in CLL cells and further regulates cell trafficking. Endothelial cells are the main source of tissue factor (TF) pathway inhibitor (TFPI), which is the primary inhibitor of TF. However, the role of TFPI in leukemic cell trafficking has not been explored before. CXCL12 and its receptor CXCR4 play an important role in CLL cell trafficking and homing. To assess the effect of TFPI on CXCL12-mediated migration of leukemic cells, we investigated transendothelial migration (TEM) of B cells from CLL patients by using CXCL12 as a chemoattractant. We found that full-length recombinant TFPI (rTFPI) increased CXCL12-mediated migration of CLL cells dose-dependently by using MACS flow cytometry to count the migrated cells. Accordingly, Western blot showed the binding of rTFPI to the CLL cells after 24 h treatment. However, the chemokine receptor CXCR4 expression in CLL cells was not increased or even slightly decreased by rTFPI treatment, suggesting that TFPI enhances leukemic cell migration probably through interacting with other factor(s) than overexpressing CXCR4 in CLL cells. Surface expression and intracellular trafficking of TFPI is regulated by glycosylphosphatidylinositol (GPI)-anchored co-receptor(s). Glypican-3, a member of proteoglycan family, binds to the plasma membrane by a GPI anchor and its heparan sulfate (HS)-type glycosaminoglycan chains are considered to mediate the interaction with other cell membrane proteins. To determine whether TFPI interacts with glypican-3, immunofluorescence staining and co-immunoprecipitation were performed in K562 and HL60 cells. Immunofluorescence analysis revealed colocalization of TFPI and glypican-3 on the surface of K562 and HL60 cells. Co-immunoprecipitation confirmed the binding between TFPI and glypican-3 in K562 cells. Moreover, co-localized TFPI and glypican-3 were found in bone marrow biopsies of leukemia patients. We further employed an antibody against HS chains of glypican-3, which inhibits the activity of glypican-3. Western blot showed that rTFPI treatment failed to increase TFPI protein in CLL cells when glypican-3 inhibitor HS20 was applied. In addition, TEM assay showed that HS20 treatment abolished the effect of TFPI on cell migration, which suggests that inhibition of glypican-3 blocks the binding of TFPI to CLL cells and then abolishes the effect of TFPI on cell migration. It indicates that TFPI affects CLL cell migration through binding to glypican-3. Using a factor Xa generation assay, we found that HS20 suppressed TF activity on the surface of HL60 cells, suggesting that the inhibition of glypican-3 impaired the procoagulant activity of leukemic cells. In addition, Western blot and RT-PCR showed that HS20 downregulated TF mRNA and protein expression in CLL cells. Finally, we explored the role of Wnt/β-catenin signaling, which has been shown to regulate tumor progression and metastasis. We found a dose-dependent increase of activated β-catenin in the CLL cells after rTFPI treatment. Inhibition of Wnt/β-catenin signaling by a potent inhibitor IWP4 strongly impaired the migration of CLL cells induced by rTFPI. In this study, we show that TFPI enhances CLL cell migration through binding to glypican-3 and activating Wnt/β-catenin signaling pathway. Our findings suggest a novel molecular mechanism regulating the transmigration of leukemic cells. It may open a new window for therapeutic targeting in the treatment of organ infiltration of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

MiR-194 regulates nasopharyngeal carcinoma progression by modulating MAP3K3 expression.

Despite the recent development of treatment strategies for nasopharyngeal carcinoma, the effective management of this disease remains a challenging clinical problem. A better understanding of the regulatory roles of miR‐194 and mitogen‐activated protein kinase kinase kinase 3 (MAP3K3) in the nasopharyngeal‐carcinoma‐related gene network is required to address this issue. Here, we measured relative expression of miR‐194 in human nasopharyngeal carcinoma tissues and normal epithelial tissues by quantitative real time PCR. We transfected cultured CNE‐1 and C666‐1 cells with miR‐194 mimics, and then examined the effects on cell proliferation, cell migration and invasion. Luciferase reporter assay was used to validate the putative binding between miR‐194 and MAP3K3. We then examined the effect of knockdown and overexpression of MAP3K3 on cell tumorigenesis. Expression of miR‐194 is significantly down‐regulated in nasopharyngeal carcinoma specimens and tumor cell lines when compared with normal controls. In addition, miR‐194 suppressed tumor cell proliferation and viability, as well as migration and invasion of carcinoma cells. We found that miR‐194 binds the 3′ untranslated region of MAP3K3, and knockdown of miR‐194 inhibited nasopharyngeal carcinoma cell proliferation, migration and invasion. In accordance, overexpression of MAP3K3 reversed the inhibitory effects of miR‐194 in carcinoma cells. This study suggests that expression of miR‐194 is down‐regulated in nasopharyngeal carcinoma, and that miR‐194 can directly target MAP3K3 to regulate tumor progression. Given the pivotal involvement of MAP3K3 in nasopharyngeal carcinoma development, targeting miR‐194 may be a novel strategy for the treatment of nasopharyngeal carcinoma.

Novel positioning from obesity to cancer: FTO, an m6A RNA demethylase, regulates tumour progression.

The fat mass- and obesity-associated (FTO) gene on chromosome 16q12.2 shows an intimate association with obesity and body mass index. Recently, research into the FTO gene and its expression product has attracted widespread interest due to the identification of FTO as an N6-methyladenosine (m6A) demethylase. FTO primarily regulates the m6A levels of downstream targets via their 3' untranslated regions. FTO not only plays a critical role in obesity-related diseases but also is involved in the occurrence, development and prognosis of many types of cancer, such as acute myeloid leukaemia, glioblastoma and breast cancer. Currently, studies indicate that FTO is a crucial component of m6A modification, it regulates cancer stem cell function, and promotes the growth, self-renewal and metastasis of cancer cells. In this review, we summarized and analysed the data regarding the structural features and biological functions of FTO as well as its association with different cancers and possible molecular mechanisms. We systematically reviewed the related literatures regarding FTO and its demethylation activity in many pathologic and physiological processes, especially in cancer-related diseases based on PubMed databases in this article. Mounting evidence indicated that FTO plays a critical role in occurrence, progression and treatment of various cancers, even acting as a cancer oncogene in acute myeloid leukaemia, research on which is no longer restricted to metabolic diseases such as obesity and diabetes. Considering FTO's critical role in many diseases, FTO may become a new promising target for the diagnosis and treatment of various diseases in the near future, especially for specific types of cancers, such as acute myeloid leukaemia, glioblastoma and breast cancer.

Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel

Architecture and microstructure of type I collagen fibers constitute central regulators of tumor invasion with aligned fibers providing a route for migration of stromal and cancer cells. Several different aspects of fibrillar collagen, such as stiffness, density, thickness, and pore size, may regulate migration of cancer cells, but determining effects of any one parameter requires clear decoupling of physical properties of collagen networks. The objective of this work is to develop and apply an in vitro three-dimensional (3D) tumor-extra cellular matrix (ECM) model with tunable physical parameters to define how stromal fibroblasts modulate collagen microstructure to control migration of breast cancer cells. We incorporated two different types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix to induce different mechanisms of gelling. The resultant biomimetic, nanocomposite hydrogels show different collagen fibrillar microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. Spheroids of human mammary fibroblasts embedded in these 3D matrices remodel the collagen network to varying extents based on differences in underlying matrix microstructures. The remodeled collagen matrix shows oriented, thicker fibrillar tracks, facilitating invasion of tumor cells. By decoupling effects of matrix stiffness and architecture, our nanocomposite hydrogels serve as robust platforms to investigate how biophysical properties of tumor environments control key processes regulating tumor progression in breast cancer and other malignancies. Statement of SignificanceOur manuscript demonstrates a new type of nanocomposite hydrogel with two different gelling mechanisms, produced by incorporating two types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix. The resultant biomimetic hydrogels show different fibrillar collagen microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. These gels allow us to uncouple effects of matrix stiffness versus architecture on migration and invasion of breast cancer cells and stromal fibroblasts. Upon embedding spheroids of human mammary fibroblasts (HMFs) and dissociated 231 breast cancer cells, we showed that HMFs remodeled the collagen network to differing extents dependent on starting matrix microstructures in each hydrogel. The remodeled collagen matrix showed aligned collagen fibers perpendicular to the surface of a spheroid with migrating HMFs following these fibers as occurs in tumors in vivo. To our knowledge, this is the first study showing significant different fibrillar collagen microstructures with constant collagen density and gel stiffness. This study establishes a new type of nanocomposite 3D hydrogels for studies of biophysical and cellular interactions in engineered tumor environments.

Pathogenic roles of the Cullin E3 ligase scaffolding protein CUL4B

<p indent=0mm>CUL4B belongs to the Cullin family, the members of which are scaffolding proteins of Cullin-RING E3 ligases (CRLs), the largest ubiquitin ligase complex in eukaryotes. CUL4B is a scaffolding protein of Cullin 4B-RING ubiquitin ligase complex (CRL4B), which catalyzes the polyubiquitination or monoubiquitination of substrate proteins and plays important roles in various physiological and pathophysiological processes. The fact that mutations in <italic>CUL4B</italic> are causally associated with human mental retardation syndrome opened a new direction for the study on the function of CUL4B. Using cell models, it was found that the CRL4B complex plays important roles in regulating cell proliferation and DNA damage repair. At the same time, it was found that CUL4B is highly expressed in various solid tumor tissues, and regulates tumor progression by epigenetically regulating the transcription of multiple tumor suppressor genes. Research using mouse models found that CUL4B played important roles in embryonic development, nervous system development, germ cell development, myeloid cell differentiation, adipocyte differentiation, and pancreatic delta cell function. This review will summarize our current knowledge of CUL4B in the pathogenesis in human diseases and mouse models.

Stromal-induced downregulation of miR-1247 promotes prostate cancer malignancy.

Cancer progression is strictly dependent on the relationship between tumor cells and the surrounding stroma, which supports cancer malignancy promoting several crucial steps of tumor progression, including the execution of the epithelial to mesenchymal transition (EMT) associated with enhancement in cell invasion, resistance to both anoikis and chemotherapeutic treatments. Recently it has been highlighted the central role of microRNAs (miRNAs) as regulators of tumor progression. Notably, in several tumors a strong deregulation of miRNAs is observed, supporting proliferation, invasion, and metabolic reprogramming of tumor cells. Here we demonstrated that cancer-associated fibroblasts induce a downregulation of miR-1247 in prostate cancer (PCa) cells. We proved that miR-1247 repression is functional for the achievement of EMT and increased cell invasion as well as stemness traits. These phenomena contribute to promote the metastatic potential of PCa cells as demonstrated by increased lung colonization in in vivo experiments. Moreover, as a consequence of miR-1247 downregulation, we observed a correlated increased expression level of neuropilin-1, a miR-1247 target involved as a coreceptor in the epidermal growth factor receptor signaling. Taken together, our data highlight miR-1247 as a potential target for molecular therapies aimed to block the progression and diffusion of PCa.

Involvement of H19/miR-326 axis in hepatocellular carcinoma development through modulating TWIST1.

Overexpression of long noncoding RNA (lncRNA) H19 has been observed in various cancers, which indicates that H19 exert important roles in the progression of carcinogenesis. MiR-326 has been reported to play tumor suppressive roles in multiple tumors. Recently, the competing endogenous RNA (ceRNA) hypothesis has implied that lncRNAs might function as molecular sponges for microRNAs in various cancers. However, the roles of H19/miR-326 in human hepatocellular carcinoma (HCC) still remain unclear. The aim of our study was to determine H19/miR-326 expression in HCC cells and investigate their roles in HCC development. We found that H19 was significantly elevated and miR-326 was decreased in HCC cells including Hep3B, HepG2, MHCC-97L, SK-hep1, Hun7, SMCC-7721 compared with LO2 cells, respectively. In the subsequent experiments, we observed that inhibition of H19 can repress HCC cell growth, migration, and invasion in vitro. H19 downregulation can increase miR-326 expression in HCC cells. Meanwhile, miR-326 mimics can also inhibit HCC progression, whereas miR-326 inhibitors exhibited a reverse phenomenon by modulating H19 expression. In addition, a negative association between H19 and miR-326 was predicted and confirmed. Furthermore, the transcription factor TWIST1 has been recognized as a significant regulator in tumor progression. Here, by performing bioinformatics analysis, TWIST1 was identified as a downstream target of miR-326. The findings of our study implied that lncRNA H19 can serve as a ceRNA to sponge miR-326 and modulate TWIST1 levels in HCC pathogenesis. Taken these together, these findings indicated that H19/miR-326/TWIST1 axis was involved in HCC development and can indicate a novel HCC target.

Increased expression of heat shock factor 1 (HSF1) is associated with poor survival in gastric cancer patients

BackgroundHeat shock factor 1 (HSF1) was initially identified as a transcription factor encoding heat shock proteins, which assist in refolding or degrading damaged proteins. Recent studies have reported that HSF1 can act as an oncogene that regulates tumour progression. The present study aimed to elucidate the clinicopathological significance and prognostic value of HSF1 expression in gastric cancer (GC).MethodsThe data from The Cancer Genome Atlas (TCGA) were used to analyse HSF1 expression in GC and normal tissues, while 8 pairs of freshly frozen tissue samples were used to investigate HSF1 expression at the mRNA and protein levels in GC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting assays. The correlations between HSF1 expression and clinicopathological parameters, including the survival rate, were investigated in 117 GC tissue samples by immunohistochemical analysis.ResultsThe results of bioinformatics analysis, qRT-PCR, and western blot showed that HSF1 expression was higher in GC tissues than in normal tissues. High HSF1 expression was found in 54.7% (64/117) patients. Patients with high HSF1 expression had larger tumour size (P = 0.001), advanced Bornmann classification (P = 0.002), advanced depth of invasion (P = 0.015), lymph node metastasis (P<0.001), distant metastasis (P = 0.011) and tumour-node-metastasis (P<0.001). Moreover, the Kaplan-Meier and Cox proportional hazards analyses indicated that high HSF1 expression was significantly associated with poor overall survival and recurrence-free survival in GC patients and that high HSF1 expression was an independent prognostic factor for the long-term survival in GC patients.ConclusionsTaken together, our results show that high HSF1 expression is significantly correlated with advanced tumour progression and poor prognosis. In addition, HSF1 expression can serve as a biomarker for the prognosis of patients with GC.

LGR5 acts as a target of miR-340-5p in the suppression of cell progression and drug resistance in breast cancer via Wnt/β-catenin pathway

Breast cancer is one of the most common malignant tumors among females. Recent studies demonstrated that microRNAs (miRNAs) played an important role in the regulation of tumor progression. In our present study, we firstly detected miR-340-5p expression in breast cancer cell lines and found lower expression of miR-340-5p in breast cancer cell lines (MCF-7, MDA-MB-231, BT-549, ZR-75-1) through qRT-PCR. Overexpressed miR-340-5p inhibited cell proliferation and drug resistance to docetaxel with enhanced cell apoptosis of breast cancer cells. Through bioinformatic prediction, we found that LGR5 was a potential target of miR-340-5p. LGR5 was highly expressed in breast cancer cells. Relative expression of LGR5 was negatively regulated by miR-340-5p. Knockdown of LGR5 also inhibited cell proliferation and drug resistance to docetaxel with enhanced cell apoptosis of breast cancer cells. Moreover, knockdown of LGR5 decreased the expression of β-catenin, c-myc, Survivin. The activation of Wnt/β-catenin pathway contracted the effects of LGR5 siRNA, indicating that LGR5 siRNA inhibited cell proliferation and drug resistance with induced apoptosis via suppressing Wnt/β-catenin signaling pathway in breast cancer. Taken together, our study demonstrated that overexpressed miR-340-5p inhibited cell proliferation and drug resistance with increased apoptosis of breast cancer cells through down-regulating LGR5 expression via Wnt/β-catenin pathway. The miR-340-5p/LGR5 axis may provide a new perspective for treatment for breast cancer.

CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cells

Circadian genes control most of the physiological functions in cancer cells, including cell proliferation, migration, and invasion. The CLOCK and BMAL1 complex plays a central role in circadian rhythms. Previous studies have shown that circadian genes may act as oncogenes or tumor-suppressor genes. In addition, F-actin, regulated by RHOA, has been shown to participate in tumor progression. However, the roles of the CLOCK and BMAL1 genes in the regulation of tumor progression via the RHOA-ROCK-CFL pathway remain largely unclear. Here we first indicate that the rearrangement of F-actin is regulated by CLOCK and BMAL1. We found that CLOCK and BMAL1 can upregulate RHOA expression by inhibiting CUL3-mediated ubiquitination and activate RHOA by reducing the interaction between RHOA and RhoGDI. Consequently, CLOCK and BMAL1 control the expression of the components of the RHOA-ROCK-CFL pathway, which alters the dynamics of F-actin/G-actin turnover and promotes cancer cell proliferation, migration, and invasion. In conclusion, our research proposes a novel insight into the role of CLOCK and BMAL1 in tumor cells.

Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efficacy of Immunotherapy in Mice.

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.Significance: These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. Cancer Res; 78(19); 5644-55. ©2018 AACR.

MiR-374b-5p-FOXP1 feedback loop regulates cell migration, epithelial-mesenchymal transition and chemosensitivity in ovarian cancer

MicroRNAs (miRNAs) are important regulators in tumorigenesis and progression of multiple human cancers, including ovarian cancer (OC). As a member of miRNAs family, miR-374b-5p has been reported to be a tumor suppressive gene in human cancers. In this study, the lower expression of miR-374b-5p was identified in OC tissues and cell liens using quantitative real time PCR (qRT-PCR). Forkhead box protein P1 (FOXP1) can act as an oncogene in human cancers. Mechanism experiments revealed that FOXP1 is a target of miR-374b-5p. Functionally, miR-374b-5p suppressed cell proliferation, migration and epithelial-mesenchymal transition (EMT) in ovarian cancer. Moreover, the sensitivity of OC cells to cisplatin was markedly enhanced by miR-374b-5p. However, FOXP1 reversed However, FOXP1 reversed miR-374b-5p-mediated biological functions. Previous reports demonstrated the inhibitory effect of FOXP1 on transcription FOXP1. Thus, we further examined the effect of FOXP1 on the transcription activity of miR-374b-5p in OC cells. The results showed that FOXP1 decreased miR-374b-5p expression by inhibiting the transcription activity of miR-374b-5p. Rescue assays revealed the regulatory effect of miR-374b-5p-FOXP1 feedback loop on ovarian cancer progression. In conclusion, miR-374b-5p-FOXP1 feedback loop regulates tumor progression and chemosensitivity in ovarian cancer.

Cancer-associated fibroblasts contribute to oral cancer cells proliferation and metastasis via exosome-mediated paracrine miR-34a-5p.

BackgroundCancer-associated fibroblasts (CAFs) play an important role in regulating tumor progression by transferring exosomes to neighboring cells. Our aim was to clarify the role of microRNA encapsulated in the exosomes derived from CAFs in oral squamous cell carcinoma (OSCC).MethodsWe examined the microRNA expression profiles of exosomes derived from CAFs and donor-matched normal fibroblasts (NFs) from patients with OSCC. We used confocal microscopy to examine the transportation of exosomal miR-34a-5p between CAFs and OSCC cells. Next, luciferase reporter and its mutant plasmids were used to confirm direct target gene of miR-34a-5p. Phenotypic assays and in vivo tumor growth experiments were used to investigate the functional significance of exosomal miR-34a-5p.FindingsWe found that the expression of miR-34a-5p in CAF-derived exosomes was significantly reduced, and fibroblasts could transfer exosomal miR-34a-5p to OSCC cells. In xenograft experiments, miR-34a-5p overexpression in CAFs could inhibit the tumorigenesis of OSCC cells. We further revealed that miR-34a-5p binds to its direct downstream target AXL to suppress OSCC cell proliferation and metastasis. Stable ectopic expression of AXL in OSCC cells overexpressing miR-34a-5p restored proliferation and motility abolished by the miRNA. The miR-34a-5p/AXL axis promoted OSCC progression via the AKT/GSK-3β/β-catenin signaling pathway, which could induce the epithelial-mesenchymal transition (EMT) to promote cancer cells metastasis. The miR-34a-5p/AXL axis enhanced nuclear translocation of β-catenin and then induced transcriptional upregulation of SNAIL, which in turn activated both MMP-2 and MMP-9.InterpretationThe miR-34a-5p/AXL axis confers aggressiveness in oral cancer cells through the AKT/GSK-3β/β-catenin/Snail signaling cascade and might represent a therapeutic target for OSCC.FundNational Natural Science Foundation of China.

Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.

Recent studies have described important roles for the sodium iodide symporter (NIS) in tumor behavior. The objectives of the present study were to investigate the role of NIS in the regulation of genes involved in tumor progression and the clinicopathological significance of its expression in gastric cancer (GC). In human GC cell lines, knockdown experiments were conducted using NIS siRNA, and the effects on proliferation, survival, and cellular movement were analyzed. The gene expression profiles of cells were examined using a microarray analysis. An immunohistochemical analysis was performed on 145 primary tumor samples obtained from GC patients. NIS was strongly expressed in MKN45 and MKN74 cells. The depletion of NIS inhibited cell proliferation, migration, and invasion and induced apoptosis. The results of the microarray analysis revealed that various interferon (IFN) signaling-related genes, such as STAT1, STAT2, IRF1, and IFIT1, were up-regulated in NIS-depleted MKN45 cells. Furthermore, the down-regulation of NIS affected the phosphorylation of MAPKs and NF-kB. Immunohistochemical staining showed that NIS was primarily located in the cytoplasm or cell membranes of carcinoma cells, and its expression was related to the histological type or venous invasion. Prognostic analyses revealed that the strong expression of NIS was associated with shorter postoperative survival. These results suggest that NIS regulates tumor progression by affecting IFN signaling, and that its strong expression is related to a worse prognosis in patients with GC. These results provide an insight into the role of NIS as a mediator and/or a biomarker for GC.

PS02.155: THE ROLE OF AQUAPORIN 1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Aquaporin 1 (AQP1) is a membrane protein whose main function is to transfer water via cellular membranes. Recent studies have described important roles for AQP1 in epithelial carcinogenesis and tumor behavior. The objectives of the present study were to investigate the role of AQP1 in the regulation of genes involved in tumor progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). Methods Immunohistochemistry for the AQP1 protein was performed to investigate the expression of AQP1 in primary tumor tissues of 50 human ESCC samples underwent curative esophagectomy. Samples were categorized into two groups according to the expression of AQP1 in each of cytoplasm and nuclear membrane, and relationships with the clinicopathological features and prognosis of ESCC patients were investigated. Next, the location of AQP1 protein in TE5, TE15, and KYSE70 cells was investigated using immunofluorescence analysis. In addition, apoptosis assay was performed in these ESCC cells and the gene expression profiles of AQP1-depleted TE5 cells was analyzed using microarray and bioinformatic studies. Results In an immunohistochemical analysis, AQP1 was primarily located in the cytoplasm and/or the nuclear membrane of carcinoma cells. The 5-year survival rate of patients with the ‘cytoplasm dominant’ expression of AQP1 (46.8%) was significantly lower than other patients (77.6%). A multivariate analysis of the 5-year overall survival rate showed that the pT categories and cytoplasm dominance groups of AQP1 were independent prognostic factors (P = 0.042 and 0.036, respectively). Immunofluorescence analysis revealed that AQP1 protein mainly existed in the cytoplasm in TE5 and TE15 cells. On the other hand, the expression of AQP1 in KYSE170 cells was confirmed on the nuclear membrane. The depletion of AQP1 using siRNA induced apoptosis in TE5 and TE15 cells. The results of microarray analysis revealed that Death receptor signaling pathway-related genes were changed in AQP1-depleted TE5 cells. Conclusion The results of the present study suggested that the cytoplasm dominant expression of AQP1 is related to a poor prognosis in patients with ESCC, and that it regulates tumor progression by affecting Death receptor signaling pathway. These results provide insights into the role of AQP1 as a biomarker for ESCC. Disclosure All authors have declared no conflicts of interest.

PS02.045: EXPRESSION AND CLINICAL SIGNIFICANCE OF LEUCINE-RICH REPEAT-CONTAINING PROTEIN 8A (LRRC8A) IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background All cells have regulatory volume decrease (RVD) function to maintain cell volume. When the cell volume changes, the anion channels on the cell membrane are activated and keep constant cell volume due to water flow. Leucine-Rich Repeat-Containing protein 8 A (LRRC8A) was first reported in 2014 as the main constituent protein of this anion channel. In normal tissues, LRRC8A is thought to control cell volume regulation and is thought to be involved in cell cycle and death. However, its expression and significance in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we examined to investigate the clinicopathological significance of its expression in ESCC and the role of LRRC8A in tumor function. Methods An immunohistochemical analysis was performed on 64 patients with ESCC who underwent curative surgery in our department. We carried out knockdown using siRNA to evaluate the role of LRRC8A in ESCC cells. Results LRRC8A was expressed in the cell membrane and cytoplasm of cancer cells. We divided ESCC patients into 2 groups by referring to the staining scores; a high expression group with staining scores ≥ 1.0, n = 53, and a low expression group with staining scores &lt; 1.0, n = 11. In correlation analysis with clinicopathologic factors, high LRRC8A expression tended to have a positive correlation with the presence or absence of lymph node metastasis (P = 0.059). In the survival analysis, the 5-year overall survival rate of patients with high LRRC8A expression (43.4%) was significantly lower than that of patients with low LRRC8A expression (90.0%) (P = 0.013). Cox proportional hazard analysis showed that high LRRC8A expression was an independent poor prognostic factor (P = 0.013, HR = 6.67). LRRC8A was strongly expressed in TE15 and KYSE170 human ESCC cells. The depletion of LRRC8A using siRNA inhibited cell proliferation, migration, invasion and induced apoptosis. Conclusion The LRRC8A expression is related to worse prognosis in ESCC patients and regulates tumor progression. Nest step, we will carry out further investigation to reveal how to regulate tumor function by microarray analysis. Disclosure All authors have declared no conflicts of interest.

Abstract B31: Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration

Abstract Macrophages are key regulators of tumor progression, and can be targeted for cancer treatment. However, reprogramming macrophages for anti-tumor activity is challenging due to their plasticity. Standard assessment of macrophage function (e.g., ELISA) lacks the sensitivity to capture cell-level heterogeneity in living macrophages. Therefore, single cell imaging and analysis tools are needed to study heterogeneous macrophage behavior within the tumor microenvironment. Macrophages adapt their metabolism to alter both phenotype and function. Thus, we propose the combination of optical metabolic imaging (OMI) and microdevice co-culture to observe cellular level macrophage heterogeneity in response to tumor stimuli. OMI enables noninvasive, dynamic imaging of intact, living samples by measuring autofluorescence from the metabolic co-enzymes NAD(P)H and FAD. Cellular level redox balance is evaluated with the optical redox ratio, defined as the ratio of NAD(P)H to FAD intensity. Additionally, NAD(P)H and FAD fluorescence lifetimes report on metabolic enzyme binding activities. Three-layer microdevices consisting of murine PyVMT breast carcinoma cells, collagen (200 micron thickness), and RAW 264.7 macrophages, respectively, were designed to monitor tumor-mediated macrophage polarization and migration. Metabolic changes with macrophage migration were measured with OMI at 24, 48, and 72 hours after culture. Redox ratio and NAD(P)H mean lifetime increased over time in co-cultured macrophages (p &amp;lt;0.05). Macrophage mono-cultures exhibited lower redox ratio and NAD(P)H lifetime (p&amp;lt;0.05) than in co-culture, consistent with the metabolism of naïve and cytokine-stimulated M2-like macrophages, respectively. Phenotype and function of co-cultured macrophages were confirmed with polymerase chain reaction (PCR) gene expression analysis and immunostaining. Furthermore, volumetric imaging visualized macrophage migration distance-dependent changes in NAD(P)H autofluorescence intensity. These results demonstrate that OMI and microdevice co-culture can capture dynamic changes in macrophage metabolism throughout 3D tumor-stimulated polarization and migration. Overall, this approach could enhance our understanding of macrophage metabolism and function, thus revealing novel therapeutic targets within the tumor microenvironment. Citation Format: Tiffany M. Heaster, Jiaquan Yu, David J. Beebe, Melissa C. Skala. Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B31.