Regulation Of Cancer Progression Research Articles

Up-regulation of circ-SMAD7 inhibits tumor proliferation and migration in esophageal squamous cell carcinoma

BackgroundIncreasing evidences demonstrate that circular RNAs (circRNAs) play an important role in the development and progression of human cancers. Nevertheless, the functions and molecular mechanism of circRNAs in esophageal squamous cell carcinoma (ESCC) tumorigenesis are largely unknown. The purpose of this research is to investigate the expression and potential role of a new circular RNA named circ-SMAD7 on ESCC carcinogenesis. Materials and methodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to measure circ-SMAD7 expression amount in both ESCC plasmas and tissues. Then the correlation between the expression of circ-SMAD7 and clinicopathological features was analyzed. Furthermore, by loss-of-function and gain-of-function experiments in ESCC cells, a functional analysis of circ-SMAD7 on ESCC cell proliferation and migration was performed. ResultsThe expression of circ-SMAD7 was validated to be significantly down-regulated in ESCC plasmas in comparison with normal controls, showing a high negative correlation with TNM stage (P = 0.000) and lymphatic metastasis (P = 0.000). Moreover, circ-SMAD7 was significantly down-regulated in ESCC tissues compared to adjacent normal tissues. Furthermore, Loss-of-function and gain-of-function experiments revealed that the expression level of circ-SMAD7 affected the proliferation and migration of ESCC cell lines. ConclusionOur study firstly exposed that over-expressioncirc-SMAD7, an influential regulator in cancer progression, can inhibit tumor proliferation and migration in ESCC and have the potential of becoming a biomarker for the diagnosis and therapy of ESCC.

Astragali radix total flavonoid synergizes cisplatin to inhibit proliferation and enhances the chemosensitivity of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the most common head and neck cancer. Astragali radix extracts play crucial roles in the regulation of cancer progression. However, the role of Astragali radix extracts in LSCC and the related mechanisms remains unclear. Here, we evaluated the inhibitory effects of the combined use of Astragali radix total flavonoid (TFA) and cisplatin (CDDP) on an LSCC mouse model by pharmacodynamics. Ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was employed to define the prototype of TFA in vivo. The potential drug targets were identified through the integrative analysis of LSCC microarrays, RNA sequencing data and the main bioactive component of TFA. Furthermore, a protein–protein interaction network, compound–target network and target–pathway network were constructed based on the prototype and potential drug targets to identify the main targets and pathways. Animal experiments showed that TFA has significant synergistic antitumor activity with cisplatin and attenuates the nephrotoxicity caused by CDDP chemotherapy, improving the survival of LSCC-bearing mice. Using UPLC-MS/MS, we identified 8 constituents of TFA in experimental mice serum: formononetin, ononin, calycosin, calycosin-7-O-β-D-glucoside, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavaneglucoside, 3-hydroxy-9,10-dimethoxypterocarpan and 9,10-dimethoxyptercarpan-3-O-β-d-glucoside. Integrative analysis predicted 19 target genes for TFA constituents, and the target genes were mainly involved in the EGFR-related cancer signaling, metabolism and oxidative stress. Collectively, these findings highlight the role of TFA in the regulation of LSCC and provide potential targets for a high-efficiency and low-toxicity therapeutic strategy of LSCC.

Knockdown of STIM1 expression inhibits non-small-cell lung cancer cell proliferation in vitro and in nude mouse xenografts

ABSTRACT Stromal interaction molecule 1 (STIM1) is a calcium-sensing protein localized in the membrane of the endoplasmic reticulum. The expression of STIM1 has been shown to be closely associated with cell proliferation. The aim of the present study was to investigate the role of STIM1 in the regulation of cancer progression and its clinical relevance. The data demonstrated that the expression of the STIM1 was significantly higher in non-small-cell lung cancer (NSCLC) tissues than in benign lesions and was associated with advanced NSCLC T stage. Knockdown of STIM1 expression in NSCLC cell lines A549 and SK-MES-1 significantly inhibited cell proliferation and induces A549 and SK-MES-1 cell arrest at the G2/M and S phases of the cell cycle. Western blotting showed that the expression of cyclin-dependent kinase (CDK) 1 and CDK2 were reduced while knockdown of STIM1 expression. Furthermore, knockdown of STIM1 in NSCLC cells significantly reduced the levels of xenograft tumor growth in nude mice. These data indicate that aberrant expression of the STIM1 protein may contribute to NSCLC progression. Future studies should focus on targeting STIM1 as a novel strategy for NSCLC therapy.

Identification of tRNA-derived ncRNAs in TCGA and NCI-60 panel cell lines and development of the public database tRFexplorer.

Next-generation sequencing is increasing our understanding and knowledge of non-coding RNAs (ncRNAs), elucidating their roles in molecular mechanisms and processes such as cell growth and development. Within such a class, tRNA-derived ncRNAs have been recently associated with gene expression regulation in cancer progression. In this paper, we characterize, for the first time, tRNA-derived ncRNAs in NCI-60. Furthermore, we assess their expression profile in The Cancer Genome Atlas (TCGA). Our comprehensive analysis allowed us to report 322 distinct tRNA-derived ncRNAs in NCI-60, categorized in tRNA-derived fragments (11 tRF-5s, 55 tRF-3s), tRNA-derived small RNAs (107 tsRNAs) and tRNA 5′ leader RNAs (149 sequences identified). In TCGA, we were able to identify 232 distinct tRNA-derived ncRNAs categorized in 53 tRF-5s, 58 tRF-3s, 63 tsRNAs and 58 5′ leader RNAs. This latter group represents an additional evidence of tRNA-derived ncRNAs originating from the 5′ leader region of precursor tRNA. We developed a public database, tRFexplorer, which provides users with the expression profile of each tRNA-derived ncRNAs in every cell line in NCI-60 as well as for each TCGA tumor type. Moreover, the system allows us to perform differential expression analyses of such fragments in TCGA, as well as correlation analyses of tRNA-derived ncRNAs expression in TCGA and NCI-60 with gene and miRNA expression in TCGA samples, in association with all omics and compound activities data available on CellMiner. Hence, the tool provides an important opportunity to investigate their potential biological roles in absence of any direct experimental evidence.Database URL: https://trfexplorer.cloud/

Circular RNA_LARP4 is lower expressed and serves as a potential biomarker of ovarian cancer prognosis.

Circular RNAs (circRNAs) have been identified as important regulators in regulatingcancerprogression. The study aims to investigate the expression of circular RNA_LARP4 (circ LARP4) and clinical significance in ovarian cancer (OC). The expression of circ LARP4 was detected in a total of 78 paired ovarian cancer tissue and adjacent normal tissue samples using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analyses. The chi-square test was used to assess the association between expression of circLARP4 and clinical-pathological parameters. Survival plot was evaluated using the Kaplan-Meier method. The multivariate Cox analysis model was used for tumor prognosis analysis. We identified that circLARP4 expression was significantly down-regulated in ovariancancertissues compared with corresponding controls. Furthermore, we found that circLARP4 expression was significantly associated with International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastases. Lower circLARP4 expression was associated with poorprognosisof OC patients. Moreover, multivariate Cox analysis showed that lower circLARP4 was an independent risk for OC prognosis. These results indicated that circLARP4 expression was lower and highlighted that circLARP4 was identified as a potential biomarker of ovarian cancer prognosis.

The roles of microRNAs related with progression and metastasis in human cancers

Metastasis is an important factor in predicting the prognosis of the patients with cancers and contributes to high cancer-related mortality. It is well-known that the EMT processes are responsible for cancer progression and metastasis. Recent studies indicated that miRNAs played a functional role in the initiation and progression of human malignancies. MicroRNAs are small non-coding RNAs of about 22 nucleotides in length that can induce mRNA degradation or repress mRNA translation by binding to the 3′UTR of their target genes. Overwhelming reports indicated that miRNAs could regulate cancer invasion and metastasis via EMT-related and/or non-EMT-related mechanisms. In this review, we concentrate on the underlying mechanisms of miRNAs in regulating cancer progression and metastasis.

Emerging role of extracellular vesicles in mediating cancer cachexia.

Cancer cachexia is a multifactorial metabolic syndrome characterized by the rapid loss of skeletal muscle mass with or without the loss of fat mass. Nearly 50-80% of all cancer patients' experience rapid weight loss results in ∼20% of cancer-related deaths. The levels of pro-inflammatory and pro-cachectic factors were significantly up-regulated in cachexia patients when compared with the patients who were without cachexia. It is becoming evident that these factors work synergistically to induce cancer cachexia. Extracellular vesicles (EVs) including exosomes and microvesicles are implicated in cell-cell communication, immune response, tissue repair, epigenetic regulation, and in various diseases including cancer. It has been reported that these EVs regulate cancer progression, metastasis, organotropism and chemoresistance. In recent times, the role of EVs in regulating cancer cachexia is beginning to unravel. The aim of this mini article is to review the recent knowledge gained in the field of EVs and cancer cachexia. Specifically, the role of tumour cell-derived EVs in promoting catabolism in distally located skeletal muscles and adipose tissue will be discussed.

Lactose-Modified Chitosan Gold(III)-PEGylated Complex-Bioconjugates: From Synthesis to Interaction with Targeted Galectin-1 Protein.

Galectins (Gal) are a family of glycan-binding proteins characterized by their affinity for β-galactosides. Galectin-1 (Gal-1), a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Coordination chemistry has been engaged to develop versatile multivalent neoglycoconjugates for binding Gal-1. In this study we report a fast and original method to synthesize hybrid gold nanoparticles in which a hydrochloridelactose-modified chitosan, named CTL, is mixed with dicarboxylic acid-terminated polyethylene glycol (PEG), leading to shell-like hybrid polymer-sugar-metal nanoparticles (CTL-PEG-AuNPs). The aim of this paper is to preliminarily study the interaction of the CTL-PEG-AuNPs with a target protein, namely, Gal-1, under specific conditions. The molecular interaction has been measured by Transmission Electron Microscopy (TEM), UV-vis, and Raman Spectroscopy on a large range of Gal-1 concentrations (from 0 to 10-12 M). We observed that the interaction was strongly dependent on the Gal-1 concentration at the surface of the gold nanoparticles.

Long noncoding RNA TUG1 promotes cell proliferation and migration of renal cell carcinoma via regulation of YAP.

Recently, long noncoding RNAs (lncRNAs) have captured much attention for their important roles in human diseases. Deregulation of lncRNA taurine-upregulated gene 1 (TUG1) has been reported to regulate cancer progression in many cancer types. However, how TUG1 contributes to renal cell carcinoma (RCC) remains elusive; we were eager to resolve the questions. Tumor tissues and the matched adjacent normal tissues were collected from patients with RCC. Messenger RNA (mRNA) levels of TUG1, yes-associated protein (YAP), and microRNA (miR)-9 levels were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The regulation of YAP by TUG1 was investigated using Western blot analysis, RT-qPCR, and immunofluorescence. The oncogenic roles of TUG1 and YAP were studied using a cell proliferation assay and a wound healing assay. The interaction of TUG1-miR-9-YAP was analyzed in RCC cell lines. In the current study, we observed a positive correlation between TUG1 expression and YAP expression in RCC using the Gene Expression Omnibus database and tumor tissues collected from 58 patients with RCC. The TUG1 elevation enhanced YAP expression but did not alter the Hippo-signaling pathway activity or YAP protein distribution in cells. In addition, we found that TUG1 could bind to miR-9; therefore, TUG1 could positively control YAP expression via downregulation of miR-9 level. Furthermore, we observed that inhibition of cell proliferation and cell migration induced by TUG1 silencing could be reversed by overexpression of YAP in RCC cell lines. Our findings indicated a pivotal role of TUG1 in driving RCC progression via regulation of miR-9/YAP, suggesting a potential therapeutic targeting role of TUG1 in RCC.

Abstract IA22: Targeting tumor-infiltrating myeloid cells for prostate cancer therapy

Abstract Prostate cancer cells are surrounded by a complex tumor microenvironment that controls prostate cancer progression and treatment resistance. We are interested in understanding the liaison between tumor-infiltrating myeloid cells and prostate cancer cells to identify new molecular mechanisms that regulate cancer progression and the emergence of castration-resistant prostate cancer (CRPC). We have previously demonstrated that myeloid-derived suppressor cells (MDSCs) trigger senescence evasion and immunosuppression in primary prostate cancers, eventually promoting tumor progression. These studies have provided the rationale to develop in the clinic novel immune therapies targeting MDSCs for the therapy of metastatic prostate cancer patients. I will present recent results on additional tumor-promoting functions played by MDSCs and tumor-associated macrophages (TAMs) in prostate cancer, and I will discuss new therapeutic strategies that combine the use of immunotherapy with androgen deprivation therapy for the treatment of CRPC Citation Format: Andrea Alimonti. Targeting tumor-infiltrating myeloid cells for prostate cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA22.

The role of ErbB3 binding protein 1 in cancer: Friend or foe?

ErbB3, a member of the epidermal growth factor receptorfamily, reportedly plays an essential role in the regulation of cancer progression and therapeutic resistance. Numerous studies have indicated that ErbB3 binding protein 1 (Ebp1), a binding partner for ErbB3, plays an important regulatory role in the expression and function of ErbB3, but there is no agreement as to whether Ebp1 also has an ErbB3-independent function in cancer and how it might contribute to tumorigenesis. In this review, we will discuss the different functions of the two Ebp1 isoforms, p48 and p42, that may be responsible for the potentially dual role of Ebp1 in cancer growth.

MEIS1 and MEIS2 Expression and Prostate Cancer Progression: A Role For HOXB13 Binding Partners in Metastatic Disease

Purpose: Germline mutations within the MEIS-interaction domain of HOXB13 have implicated a critical function for MEIS-HOX interactions in prostate cancer etiology and progression. The functional and predictive role of changes in MEIS expression within prostate tumor progression, however, remain largely unexplored.Experimental Design: Here we utilize RNA expression datasets, annotated tissue microarrays, and cell-based functional assays to investigate the role of MEIS1 and MEIS2 in prostate cancer and metastatic progression.Results: These analyses demonstrate a stepwise decrease in the expression of both MEIS1 and MEIS2 from benign epithelia, to primary tumor, to metastatic tissues. Positive expression of MEIS proteins in primary tumors, however, is associated with a lower hazard of clinical metastasis (HR = 0.28) after multivariable analysis. Pathway and gene set enrichment analyses identified MEIS-associated networks involved in cMYC signaling, cellular proliferation, motility, and local tumor environment. Depletion of MEIS1 and MEIS2 resulted in increased tumor growth over time in vivo, and decreased MEIS expression in both patient-derived tumors and MEIS-depleted cell lines was associated with increased expression of the protumorigenic genes cMYC and CD142, and decreased expression of AXIN2, FN1, ROCK1, SERPINE2, SNAI2, and TGFβ2.Conclusions: These data implicate a functional role for MEIS proteins in regulating cancer progression, and support a hypothesis whereby tumor expression of MEIS1 and MEIS2 expression confers a more indolent prostate cancer phenotype, with a decreased propensity for metastatic progression. Clin Cancer Res; 24(15); 3668-80. ©2018 AACR.

Tescalcin/c-Src/IGF1R\u03b2-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

Tescalcin (TESC; also known as calcineurin B homologous protein 3, CHP3) has recently reported as a regulator of cancer progression. Here, we showed that the elevation of TESC in non-small cell lung cancer (NSCLC) intensifies epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, consequently enhancing the cellular resistance to γ-radiation. TESC expression and the phosphorylation (consequent activation) of signal transducer and activator of transcription 3 (STAT3) were upregulated in CSC-like ALDH1high cells than in ALDH1low cells sorted from A549 NSCLC cells. Knockdown of TESC suppressed CSC-like properties as well as STAT3 activation through inhibition of insulin-like growth factor 1 receptor (IGF1R), a major signaling pathway of lung cancer stem cells. TESC activated IGF1R by the direct recruitment of proto-oncogene tyrosine kinase c-Src (c-Src) to IGF1Rβ complex. Treatment of IGF1R inhibitor, AG1024, also suppressed c-Src activation, implicating that TESC mediates the mutual activation of c-Src and IGF1R. STAT3 activation by TESC/c-Src/IGF1R signaling pathway subsequently upregulated ALDH1 expression, which enhanced EMT-associated CSC-like properties. Chromatin immunoprecipitation and luciferase assay demonstrated that STAT3 is a potential transcription activator of ALDH1 isozymes. Ultimately, targeting TESC can be a potential strategy to overcome therapeutic resistance in NSCLC caused by augmented EMT and self-renewal capacity.

Tumor endothelial marker 8 promotes cancer progression and metastasis

Every year more than 8 million people suffer from cancer-related deaths worldwide [1]. Metastasis, the spread of cancer to distant sites, accounts for 90% of these deaths. A promising target for blocking tumor progression, without causing severe side effects [2], is Tumor Endothelial Marker 8 (TEM8), an integrin-like cell surface protein expressed predominantly in the tumor endothelium and in cancer cells [3, 4]. Here, we have investigated the role of TEM8 in cancer progression, angiogenesis and metastasis in invasive breast cancer, and validated the main findings and important results in colorectal cancer. We show that the loss of TEM8 in cancer cells results in inhibition of cancer progression, reduction in tumor angiogenesis and reduced metastatic burden in breast cancer mouse models. Furthermore, we show that TEM8 regulates cancer progression by affecting the expression levels of cell cycle-related genes. Taken together, our findings may have broad clinical and therapeutic potential for breast and colorectal primary tumor and metastasis treatment by targeting TEM8.

Chemokine (C-X-C motif) ligand 1 is associated with tumor progression and poor prognosis in patients with colorectal cancer.

Chemokine (C-X-C motif) ligand 1 (CXCL1) is a chemotactic cytokine known to regulate cancer progression and invasion. However, the prognostic significance of CXCL1 expression in colorectal cancer (CRC) has not been fully characterized. The present study explored the clinicopathological significance and potential role of CXCL1 in the carcinogenesis and progression of CRC. The protein expression of CXCL1 was measured immunohistochemically in tissue microarrays constructed from 276 CRC patients. CXCL1 expression levels and their associations with clinicopathological characteristics and patient survival were evaluated. The effect of CXCL1 on glycolysis was also examined. High CXCL1 expression was detected in 165 (59.8%) cases. CXCL1 expression was correlated with tumor diameter (P=0.002), T stage (P=0.044), N stage (P=0.005), M stage (P=0.001), lymphovascular invasion (P=0.010), and carcinoembryonic antigen status (P=0.019). High CXCL1 expression was validated as an independent prognostic factor for overall survival (OS) and disease-free survival (DFS) by both univariate and multivariate Cox regression analyses (both P<0.05). Experimentally, expression of CXCL1 was knocked down by stable transfected short hairpin RNA, resulting in a significantly decreased rate of glycolysis both in in vitro assays and in patients’ samples (P<0.05). Silencing the expression of CXCL1 decreased the levels of the glycolytic enzymes GLUT1, HK2, and LDHA. In conclusion, by inducing glycolysis, CXCL1 plays a crucial role in both cancer progression and metastasis in CRC patients. The CXCL1 expression level is an independent prognostic factor for both OS and DFS. Moreover, CXCL1 may serve as a new biomarker and potential therapeutic target for CRC treatment.

Abstract 1111: Role of a prometastatic miRNA as a negative regulator of the key metastasis suppressor genes in renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is among the ten leading cancer types in United States. The 5-year relative survival rate of regional or localized RCC is 65-92%, while that of metastatic RCC is only 12%. Tumor recurrence and metastasis represent two major obstacles in the successful treatment of cancer. Emerging lines of evidence suggest that cancer aggressiveness is associated with epithelial to mesenchymal transition (EMT). Therefore, it is of critical importance to regulate EMT and to develop effective therapeutic strategies for the treatment of recurrent and metastatic cancer. Critical regulators of EMT include transcription repressors and microRNAs that target key proteins involved in EMT. MicroRNAs are implicated in regulating cancer progression and metastasis. Here we show that miR-720 is positively associated with RCC by negatively regulating key metastasis suppressing genes. Methods: We performed a series of in vitro and in vivo experiments including qRT-PCR, FACS cell cycle and apoptosis, chemotactic transwell migration and invasion, immunoblotting and luciferase reporter assays along with intratumoral xenograft mouse model. Results: Elevated levels of miR-720 were observed in a panel of RCC cell lines and clinical tissues compared to non-malignant cell line and normal samples. Loss of miR-720 function inhibited proliferation, migration and invasion and induced apoptosis in RCC cell lines in vitro and repressed tumor growth in xenograft mouse model. Conversely, gain of miR-720 function in non-malignant HK-2 cells induced pro-cancerous characteristics. Silencing of miR-720 caused a marked induction in the levels of endogenous αE-catenin and E-cadherin protein levels in anti720 transfected cells compared to control. Whereas, miR-720 overexpression in RCC cell lines reduced activity of a luciferase reporter gene fused to the wild-type αE-catenin or E-cadherin 3' UTR compared to non-specific 3' UTR control indicating that αE-catenin-E-cadherin complex is a direct and functional target of miR-720 in RCC. We also observed attenuation of β-Catenin, CD44 and Akt expression in RCC cells transfected with miR-720 inhibitor compared to control. Further, miR-720 exhibited clinical significance in RCC. Expression of miR-720 significantly distinguished malignant from normal samples. Elevated miR-720 levels positively correlated with higher Fuhrman grade, pathological stage and poor overall survival of RCC patients. Conclusion: These findings uncover a new regulatory network in RCC involving metastasis-promoting miR-720 that directly targets expression of key metastasis-suppressing proteins E-cadherin and αE-catenin complex. These results suggest that therapeutic regulation of miR-720 may provide an opportunity to regulate EMT and metastasis in RCC. Citation Format: Nadeem S. Bhat, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Priyanka Kulkarni, Pritha Dasgupta, Z Laura Tabatabai, Guoren Deng, Rajvir Dahiya, Shahana Majid. Role of a prometastatic miRNA as a negative regulator of the key metastasis suppressor genes in renal cell carcinoma [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 1111.

Abstract 1738: PD-1/PD-L1 expression and tumor-infiltrating immune cells in triple-negative breast cancer: Characterization of preclinical primary tumor and bone metastasis models in humanized mice

Abstract Expression of programmed cell death-1 receptor (PD-1), programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown in many studies supporting development of immunotherapies against TNBC. In order to develop novel immunotherapies especially against bone metastatic TNBC, better understanding of PD-1/PD-L1 axis, TILs, and tumor associated macrophages (TAMs) in the bone microenvironment is warranted. Aim of this study was to characterize immune cell reaction between TNBC primary tumor and bone metastasis in humanized mice to support immunotherapy drug discovery against bone metastasis. MDA-MB-231(SA) human breast carcinoma cells were inoculated into mammary fat pad or tibia of female CIEA NOG mice engrafted with human pluripotent CD34+ cells and tumor growth was followed for 3 weeks. Immunohistochemical stainings of primary tumors and intratibial tumors were performed against human specific antigens to characterize expression of PD-1, PD-L1, Granzyme B, CD4, CD8 and CD68/CD163. TILs and TAMs were assessed by 4-scale immunoscoring system and PD-L1 expression was determined by Tumor Proportion Scoring (TPS). PD-L1 positivity of the TAM's was omitted in TPS interpretation. Carcinoma cells of orthotopical tumors exhibited mainly low or moderate heterogeneous expression of PD-L1 (TPS score 1-49%). Moderate or high numbers of CD4 and CD8 positive TILs (scoring 2-3) and low PD-1 expression were observed. Granzyme B expression correlated with CD8 positivity. In comparison, when MDA-MB-231(SA) carcinoma cells were inoculated into bone marrow, tumors had corresponding PD-L1 expression and tumor-infiltrating CD4+ and granzyme B+ human immune cells were observed. However, in the bone microenvironment less CD8+ immune cells were observed and PD-1 expression was negative. When analyzing all tumor area, intratumoral and peritumoral variation of the marker expression and cell location of TILs and TAMs was observed especially in the bone tumors but also in the primary tumors. TILs and TAMs occurred in scattered as well as aggregate form in tumor area. Taken together, TNBC primary tumors and bone metastases had mainly low or moderate, but clear membranous expression of PD-L1. TILs consisted of CD4+, CD8+, and granzyme B+ cells in the primary tumors, but less CD8+ cells were observed in the bone metastases. PD-1 expression was negative in bone metastases. The obtained results comparing primary tumor and bone metastasis highlights the importance of understanding the influence of tumor microenvironment. Immune system is not only strong regulator of cancer progression, but it also regulates bone turnover. Therefore, it is crucial to use predictive preclinical models when assessing novel immunotherapies. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Azusa Tanaka, Michael Seiler, Teppo Haapaniemi, Jenni Bernoulli. PD-1/PD-L1 expression and tumor-infiltrating immune cells in triple-negative breast cancer: Characterization of preclinical primary tumor and bone metastasis models in humanized mice [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 1738.

Abstract 2918: Decursin from Angelica gigas Nakai regulates CXCR7 expression and its downstream signaling in gastric cancer cells

Abstract Background: C-X-C motif chemokine receptor 7 (CXCR7) is a member of the G protein-coupled receptor family and is reported to be involved in the invasion, migration and tumor progression of various cancers. However, few compounds regulate CXCR7 expression. Therefore, compounds that negatively regulate CXCR7 may inhibit tumor progression and thus serve as candidates for effective anti-cancer drugs. In this study, we investigated the regulation of CXCR7 by decursin derived from Angelica gigas and its downstream signaling. Methods: A CXCR7-overexpressing stable cell line was established in gastric cancer cells, and the expression of membrane CXCR7 was confirmed by flow cytometry and immunocytochemistry. Cell viability was examined using the CCK-8 assay. We investigated the potential of decursin to inhibit cell migration and invasion using a Transwell system. We also examined the mechanism of CXCR7 regulation by decursin by performing Western blot analysis of downstream proteins using phospho-specific antibodies. Results: CXCR7 overexpression significantly increased the proliferation, invasion and migration of gastric cancer cells. In addition, spheres were well formed under anchorage-independent conditions in CXCR7-expressing gastric cancer cells. Levels of phospho-Stat3 and -Akt, positive regulators of cancer progression, were significantly increased. Decursin treatment reduced CXCR7 expression at the cellular membrane, and significantly decreased cancer cell invasion and migration. When treated with decursin, the sphere formation capacity decreased in a dose-dependent manner. In addition, decursin decreased the phosphorylation of Stat3 and its downstream target, c-Myc; however, it had no effect on Akt phosphorylation. Conclusion: Decursin reduces the invasion, migration and tumor progression of cancer cells by inhibiting CXCR7. Decursin could be proposed as a new anti-cancer drug by decreasing the expression of CXCR7, which is believed to be a therapeutic target in gastric cancer. Citation Format: Solbi Kim, Nayoung Kim, Hyewon Ryu, Gyu-Yong Song, Hyo Jin Lee. Decursin from Angelica gigas Nakai regulates CXCR7 expression and its downstream signaling in gastric cancer cells [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 2918.

Abstract 494: Intravital discovery of miRNA drivers of human cancer cell directional invasion

Abstract Metastatic cancer cells use directional ECM cues such as blood vessels or collagen fibers when invading through live tissue. Oncogenic miRNAs have been implicated as key regulators of cancer progression, yet the systemic discovery of miRNAs that drive directional cancer cell invasion has not been achieved. Here we describe the first in vivo quantitative whole human miRNAome screen for miRNA drivers of directional cancer cell invasion that is based on high-resolution intravital imaging. We identified several novel miRNAs that promote cancer cell invasion during the key rate-limiting step of cancer metastasis: the initiation of overt metastatic lesions. In vivo 4D cancer cell tracking revealed that these prometastatic miRNAs are required for successful invasion into collagen-rich tissue and for attachment to the outer surface of the vascular wall. Deregulation of these miRNAs led to formation of loose contacts with the vasculature and chaotic, nondirectional cancer cell invasion patterns in living tissue. Intravital SHG microscopy showed that inhibition of the expression of these miRNAs blocked the ability of cancer cells to rearrange the disorganized collagen network into collagen fiber bundles and stably protrude along these bundles. Further imaging analysis revealed that this miRNA expression specifically blocks vesicular transport of key cell invasion machinery proteins such as MT1-MMP to the cancer cell invadopodia. Human cancer gene expression database analysis showed that our top miRNA candidates are specifically deregulated in invasive pancreatic cancer. Indeed, intravital imaging analysis showed that blocking of metastatic miRNA function inhibited MT1-MMP secretion and ECM degradation by human pancreatic cancer cells. In summary, we identified a novel panel of human miRNAs that are functionally involved in the regulation of directional invasion and metastasis. This work establishes these miRNAs as promising therapeutic targets to block the metastatic spread of lethal cancers. Citation Format: Konstantin V. Stoletov, Lian Willetts, Juan Jovel, Emma Woolner, John D. Lewis. Intravital discovery of miRNA drivers of human cancer cell directional invasion [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 494.

PO-284 Hypoxia mediates cancer stem cell (CSC) enrichment through downregulation of oestrogen receptor alpha (ERα) expression in ER positive breast cancer cells

IntroductionCSCs are a subpopulation of cancer cells which are responsible for tumour initiation and progression. It is well known that hypoxia regulates cancer progression by upregulating CSC phenotype. However, molecular mechanism by which hypoxia regulates CSC phenotype is yet to be elucidated. In our study, we proposed a novel mechanism by which hypoxia regulates CSC phenotype in breast cancer cells through regulation of ERα.Material and methodsThe effect of hypoxia on regulation of breast CSC phenotype and epithelial-mesenchymal transition (EMT) marker expression was examined in MCF-7 cells using various in vitro assays. The expression of ERα was analysed by using Immunoblotting and qRT-PCR. CSC and non-CSC populations were sorted by FACS using breast CSC-specific markers. Role of ERα in regulation of CSC phenotype was evaluated by using ERα specific siRNA. Effect of selective oestrogen receptor modulators (SERMs) in regulating CSC marker expression was assessed by immunoblotting.Results and discussionsWe observed that hypoxia enriches CSC phenotype and upregulates EMT marker expression by targeting the expression of ERα. CSC phenotype negatively correlates with ERα status of breast cancer cells. Isolated CSCs express low levels of ERα compared to non-CSCs. Hypoxia inducible factor 1 alpha (HIF1α) downregulates the expression of ERα under hypoxic condition, that leads to the enrichment of CSC population in ER positive breast cancer cells. Interestingly, knockdown of ERα by specific siRNA enriches CSC population in normoxic condition. Treatment of breast cancer cells with SERMs upregulates the expression of EMT specific genes and CSC specific transcription factors in ER positive breast cancer cells.ConclusionThese findings suggest that HIF1α downregulates ERα expression under hypoxic condition. Downregulation of ERα upregulates CSC phenotype and promotes EMT in breast cancer cells. Treating breast cancer cells by targeting ERα using SERMs may contribute to therapeutic resistance by enriching CSC phenotype.