Promotion Of Epithelial-mesenchymal Transition Research Articles

Crucial relationship of Neural Wiskott Aldrich syndrome protein(N-WASP) and Lysyl oxidase-like 2(LOXL2) in the promotion of pancreatic cancer metastasis

Introduction: Pancreatic cancer has been widely known to have aggressive malignancy and poor prognosis. N-WASP is part of the WASP family that regulate actin polymerization by activating Arp2/3 complex. FAK(Focal adhesion kinase) promotes N-WASP and epithelial mesenchymal transition(EMT) in cancer metastasis. The overexpression of LOXL2 promotes distant metastasis in pancreatic cancer. However, there is insufficient evidence of the relationship between LOXL2 and N-WASP in pancreatic cancer. In this study, we aimed to confirm the expression of N-WASP and relation between LOXL2 and N-WASP in pancreatic cancer. Method: Between June 2002 and December 2012, 81 patients underwent radical curative resection for pancreatic cancer at Gangnam Severance Hospital. Pancreatic cancer cell lines MIA PaCa-2, PANC-1 were used for in vitro study. To evaluate the endogenous expression level of N-WASP, we purified the whole RNA and protein to perform the qPCR, RT-PCR and Western blot. And we confirmed the motility and invasiveness of MIA PaCa-2 and PANC-1. Result: High expression of LOXL2 and N-WASP was observed and correlated with the expression of mesenchymal markers (Snail, L1CAM, Vimentin). We generated stably LOXL2 silenced pancreatic cancer cell which exhibited significant changes of factors related to invasiveness and N-WASP expression. Importantly, silencing of N-WASP markedly decrease in motility and invasiveness of MIA PaCa-2 and PANC-1 as determined from wound healing assay. Conclusion: Our results demonstrated that N-WASP is a regulator of EMT for migration and invasion in pancreatic cancer. LOXL2 and N-WASP have a powerful relationship for activating the promotion of EMT in pancreatic cancer metastasis. These findings suggest that N-WASP can be a target for the determining of distant metastasis in pancreatic cancer.

Claudin-7 downregulation induces metastasis and invasion in colorectal cancer via the promotion of epithelial-mesenchymal transition

The dysregulation of the tight junctions (TJs) protein claudin-7 is closely related to the development and metastasis of colorectal cancer (CRC). The aim of this study was to investigate the expression of claudin-7 and characterize the relationship between claudin-7 expression and epithelial-mesenchymal transition (EMT) in CRC. In this study, the expression of claudin-7, E-cadherin, vimentin and snail-1 was detected by immunohistochemistry (IHC) in a set of 80 CRC specimens comprising 20 specimens each of well-differentiated, moderately differentiated, poorly differentiated and liver metastases tissues. The correlation between claudin-7 and EMT-related proteins in the stably transfected claudin-7 knockdown HCT116 cell line was analyzed by IHC, immunofluorescence (IF), Western blotting (WB) and nude mouse xenograft models. The results revealed that the expression of claudin-7 was downregulated as CRC tissue differentiation grade decreased, and that low claudin-7 expression corresponded to the downregulation of E-cadherin (r = 0.725, p < 0.001) and upregulation of vimentin (r = −0.376, p = 0.001) and snail-1 (r = −0.599, p < 0.001). Additionally, in the claudin-7 knockdown HCT116 cell line, the staining intensity and expression of E-cadherin was decreased, while the immunoreactivity and expression of vimentin and snail-1 was increased. Futhermore, the result of tumor formation experiment was consistent with CRC tissues. In conclusion, the expression of claudin-7 in CRC is downregulated as differentiation grade decreases. Claudin-7 downregulation may promote the invasion and metastasis of CRC by regulating EMT. Our results provide new perspectives for a potential therapeutic target for CRC.

Activated hepatic stellate cells promote epithelial-to-mesenchymal transition in hepatocellular carcinoma through transglutaminase 2-induced pseudohypoxia

Activation of hepatic stellate cells reportedly contributes to progression of hepatocellular carcinoma (HCC). Herein, we use quantitative proteomics and ingenuity pathway analysis to show that transglutaminase 2 (TGM2) is upregulated in the course of activated hepatic stellate cells promoting epithelial-mesenchymal transition (EMT) in HCC-derived cells both in vivo and in vitro. Mechanistically, activated hepatic stellate cells promote TGM2 upregulation in HCC cells through inflammatory signalling; and TGM2-induced depletion of von Hippel-Lindau (VHL) protein, a key molecule in the degradation of hypoxia inducible factor-1a (HIF-1a) under normoxia, then causes HIF-1a to accumulate, thereby producing a pseudohypoxic state that promotes EMT in HCC cells. These findings suggest that the promotion of EMT in HCC cells by activated hepatic stellate cells is mediated by pseudohypoxia induced via TGM2/VHL/HIF-1a pathway.

Optogenetic control of epithelial-mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of cancer cell metastasis. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to be involved in TGF-β induced EMT, but the complicated TGF-β signaling network makes it challenging to dissect the important role of PI3K on regulation of EMT process. Here, we applied optogenetic controlled PI3K module (named ‘Opto-PI3K’), which based on CRY2 and the N-terminal of CIB1 (CIBN), to rapidly and reversibly control the endogenous PI3K activity in cancer cells with light. By precisely modulating the kinetics of PI3K activation, we found that E-cadherin is an important downstream target of PI3K signaling. Compared with TGF-β treatment, Opto-PI3K had more potent effect in down-regulation of E-cadherin expression, which was demonstrated to be regulated in a light dose-dependent manner. Surprisingly, sustained PI3K activation induced partial EMT state in A549 cells that is highly reversible. Furthermore, we demonstrated that Opto-PI3K only partially mimicked TGF-β effects on promotion of cell migration in vitro. These results reveal the importance of PI3K signaling in TGF-β induced EMT, suggesting other TGF-β regulated signaling pathways are necessary for the full and irreversible promotion of EMT in cancer cells. In addition, our study implicates the great promise of optogenetics in cancer research for mapping input-output relationships in oncogenic pathways.

MiR-98 modulates macrophage polarization and suppresses the effects of tumor-associated macrophages on promoting invasion and epithelial\u2013mesenchymal transition of hepatocellular carcinoma

BackgroundTumor-associated macrophages (TAMs) are generally recognized as a promoter of tumor progression. miR-98 has been shown to suppress the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells. Here, we aim to investigate the role of miR-98-mediated macrophage polarization in HCC progression.MethodsHuman blood monocytes were isolated from healthy male donors and incubated with culture medium collected from HepG2 cells for 7 days. The phenotype of the macrophages was detected. The protein expression was detected by Western blot. Levels of cytokines secreted in culture medium were measured using the specific enzyme-linked immunosorbent assay kits. To explore the role of miR-98 in HCC-conditioned TAMs, HCC cells HepG2 and SMMC7721 were cultured with conditioned medium from HCC-conditioned TAMs that had been transfected with miR-98 mimic/inhibitor. Cell proliferation, migration and invasion assays were performed.ResultsHCC-conditioned TAMs possessed M2-like phenotype, including increased protein expression of CD163 and TNF-αlow, IL-1βlow, TGF-βhigh and IL-10high phenotype. HCC-conditioned TAMs also promoted proliferation, migration, invasion and EMT of HepG2 and SMMC7721 cells. Furthermore, miR-98 modulated macrophage polarization from M2 to M1 in HCC-conditioned TAMs, as evidenced by the alteration of M1- or M2-related cytokines. Moreover, miR-98 mimic significantly suppressed the HCC-conditioned TAMs-mediated promotion of cell migration, invasion and EMT in HepG2 and SMMC7721 cells compared with negative control, whereas miR-98 inhibitor exerted reversed effects.ConclusionsmiR-98 may play a vital role in regulating macrophage polarization, thereby suppressing the TAMs-mediated promotion of invasion and EMT in HCC.

Abstract 2875: Calycosin is a double-edged sword in the treatment of pancreatic cancer

Abstract Calycosin is known for its suppression of breast cancer cell growth. We are the first group to report the effects of calycosin in pancreatic cancer development. Growth inhibition of the human pancreatic cell line MIA PaCa-2 was evaluated by MTT assay. Cell cycle distribution was determined by flow cytometry. Cell migratory activity was investigated by a wound healing assay. Subcutaneous xenografting of MIA PaCa-2 cells was conducted in Balb/C nude mice, while orthotopic inoculation of the mouse Pan01 pancreatic cancer cells was performed in C57BL/6 mice. Our results have shown that calycosin exhibited modest growth inhibition of MIA PaCa-2 cells, together with induction of S phase arrest and restoration of p53. Besides, promotion of both caspase-dependent apoptosis and autophagy implicate that the anticancer mechanism of calycosin strongly rely on programmed cell death. Alternatively, calycosin also inhibited the migration of MIA PaCa-2 cells in line with downregulation of epithelial–mesenchymal transition (EMT) and pro-metastatic biomarkers. The above in vitro benefits of calycosin was confirmed by a significant reduction of tumor mass in the MIA PaCa-2 xenograft. Nevertheless, when we look closely on the in vivo anticancer effect of calycosin in the Pan01 orthotopic mice model, although both attenuation of tumor growth and proapoptotic action were confirmed by histological findings, the tumor tissue expression of Snail, vimentin and CD31 was all elevated, indicating possible promotion of EMT and pro-angiogenic potential of the drug. These controversial findings suggest that calycosin could be an effective anticancer agent by promoting programmed cell death and inhibition of cell proliferation of pancreatic cancer cells, while it may also facilitate metastatic development of tumor, particular at the advanced stages. It would be essential to further dissect the conditions and factors that differentiate the dual actions of calycosin in pancreatic cancer therapy. Citation Format: Zhu Zhang, Joshua KS Ko. Calycosin is a double-edged sword in the treatment of pancreatic 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 2875.

RETRACTED ARTICLE: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

BackgroundA deeper mechanistic understanding of epithelial-to-mesenchymal transition (EMT) regulation is needed to improve current anti-metastasis strategies in ovarian cancer (OvCa). This study was designed to investigate the role of lncRNAs in EMT regulation during process of invasion-metastasis in serous OvCa to improve current anti-metastasis strategies for OvCa.MethodsWe systematically analyzes high-throughput gene expression profiles of both lncRNAs and protein-coding genes in OvCa samples with integrated epithelial (iE) subtype and integrated mesenchymal (iM) subtype labels. Mouse models, cytobiology, molecular biology assays and clinical samples were performed to elucidate the function and underlying mechanisms of lncRNA PTAF-mediated promotion of EMT and invasion-metastasis in serous OvCa.ResultsWe constructed a lncRNA-mediated competing endogenous RNA (ceRNA) regulatory network that affects the expression of many EMT-related protein-coding genes in mesenchymal OvCa. Using a combination of in vitro and in vivo studies, we provided evidence that the lncRNA PTAF-miR-25-SNAI2 axis controlled EMT in OvCa. Our results revealed that up-regulated PTAF induced elevated SNAI2 expression by competitively binding to miR-25, which in turn promoted OvCa cell EMT and invasion. Moreover, we found that silencing of PTAF inhibited tumor progression and metastasis in an orthotopic mouse model of OvCa. We then observed a significant correlation between PTAF expression and EMT markers in OvCa patients.ConclusionsThe lncRNA PTAF, a mediator of TGF-β signaling, can predispose OvCa patients to metastases and may serve as a potential target for anti-metastatic therapies for mesenchymal OvCa patients.

Trop2 is a potential biomarker for the promotion of EMT in human breast cancer.

Trop2 is considered to have an important function in tumor metastasis and the promotion of epithelial‑mesenchymal transition (EMT). E‑cadherin is a crucial factor in intercellular adhesion and EMT transformation. In the present study, we detected the expression of Trop2 and E‑cadherin in breast cancer (BC) to better define their prognostic value. The mRNA expression levels of these two genes in 20cases of fresh BC tissues were detected by quantitative real‑time polymerase chain reaction (qRT‑PCR). We also detected the expression levels of these two genes by immunohistochemistry (IHC) in 312BC tissues, and the correlations between the expression of these two genes and the clinicopathological characteristics in BC patients were analyzed. The mRNA and protein expression levels of the two genes in BC cell lines were studied by qRT‑PCR and western blotting. The results indicated that Trop2+/E‑cadherin‑ was expressed in BC tissues more than that in the matched adjacent tissues. The protein expression results obtained via IHC were similar to the mRNA expression results. Trop2+/E‑cadherin‑ that was expressed in BC was associated with lymph node status, metastasis, tumor‑node‑metastasis (TNM) stage, and ER‑/PR‑/HER2‑ expression. BC patients that expressed Trop2+/E‑cadherin‑ had poor overall survival rates. The results of Trop2 and E‑cadherin expression levels obtained in the BC cell lines were the same as those obtained in the BC tissues. Overall, Trop2 has a potential role in the promotion of EMT in BC and it could be considered as a therapeutic target in the future.

TIMP‐2 Inhibits Triple Negative Breast Cancer Growth and Metastasis through EMT Suppression and Promotion of Vascular Normalization

Successful tumor cell invasion requires degradation of the extracellular matrix (ECM) by matrix metalloproteases (MMPs), a large family of metalloproteinases that cleave ECM proteins. MMP synthesis and activation is tightly regulated by their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). TIMP‐2 is a multifunctional protein shown to inhibit tumor growth, invasion and metastasis independent of MMP inhibitory activity via mechanisms including alteration of the epithelial to mesenchymal transition (EMT), reduced tumor angiogenesis, and inhibition of VEGF‐A induced vascular permeability. Triple‐negative breast cancers (TNBCs) are highly vascular tumors that exhibit EMT plasticity and extensive metastatic dissemination. We explored the efficacy of recombinant TIMP‐2 (rTIMP‐2) as a preclinical therapeutic using an orthotopic mouse model which phenotypically resembles human TNBC and establishes spontaneous lung metastases. Daily rTIMP‐2 treatment (100 ug/kg/day) was initiated 10 days post tumor cell implantation and continued through day 37. Biweekly tumor measurements [tumor volume) = 1/2(length × width2)] revealed reduced primary tumor growth in rTIMP‐2 treated mice 15 days post inoculation and maximum tumor growth inhibition on day 29. Immunostaining of primary tumors demonstrated increased CK‐18 expression along with decreased CK‐14 and vimentin in rTIMP‐2 treated mice, suggesting reversal of the EMT phenotype by day 32, while lung histology showed a significant reduction in metastatic foci in rTIMP‐2 treated mice at day 52. Moreover, immunofluorescence demonstrates increased pericyte coverage and reduced vascular leakage in rTIMP‐2 treated primary TNBC tumors, showing modulation of vessel morphology and promotion of events consistent with vessel normalization. These findings suggest the therapeutic potential for rTIMP‐2 in the clinical treatment of TNBC.Support or Funding InformationThis work was funded by the NCI Center for Cancer Research Intramural Research Program Project # ZIA SC009179‐25.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FSCN‑1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition.

Resistance to chemotherapy drugs remains a significant problem for the treatment of many types of cancer. Fascin-1 (FSCN-1) is an actin-bundling protein involved in the invasion and metastasis of a variety of tumors. However, its involvement in drug resistance in hepatocellular carcinoma (HCC) remains unclear. The present study aimed to investigate the function of FSCN-1 in HCC resistance to doxorubicin (DOX). FSCN-1 expression was increased in DOX-resistant HCC cell lines (SNU449 and SNU387) compared with DOX-sensitive cell lines (Huh7 and Hep3B). The resistance of HCC cells to DOX was decreased following FSCN-1 knockdown with small interfering RNA. FSCN-1 knockdown also significantly altered the expression of key markers of epithelial-mesenchymal transition (EMT). Notably, vimentin expression was reduced and epithelial-cadherin expression was increased. Furthermore, when EMT was suppressed through knockdown of Twist, an essential pathway of DOX-induced EMT, the viability of HCC cells following treatment with DOX was not affected by FSCN-1 expression. Furthermore, FSCN-1 knockdown eliminated hypoxia-induced doxorubicin resistance and EMT. The results of the present study indicated that FSCN-1 expression increased DOX resistance in HCC cells via the promotion of EMT, and this phenomenon was maintained in a hypoxic environment. FSCN-1 potentially represents a novel target to overcome resistance to DOX in HCC.

Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail

Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail.

Prognostic Significance of Zinc Finger E-Box-Binding Homeobox Family in Glioblastoma.

BackgroundEpithelial-mesenchymal transition (EMT) is an essential progress for tumor cell invasion to both epithelial and non-epithelial cancers, and zinc finger E-box-binding homeobox 1/2 (ZEB1/2) is a well-known promoter of EMT. In glioma cell lines, both ZEB1 and ZEB2 have been demonstrated to facilitate cancer cell proliferation and invasion with experiments in vitro. However, the clinical significance of ZEB1 and ZEB2 in glioblastoma (GBM) is still controversial.Material/MethodsWe detected the expression of ZEB1 and ZEB2 in 91 cases of GBM with immunohistochemistry and investigated the correlation between clinicopathological factors and ZEB family expression with Fisher test. By univariate analysis with Kaplan-Meier test, we explored the prognostic significance of ZEB1/2 expression and the clinicopathological factors in GBM. By multivariate analysis with the Cox regression model, we identified the independent prognostic factors in GBM.ResultsThe percentages of ZEB1 high expression and ZEB2 high expression were 31.9% (29/91) and 41.9% (36/91), respectively. High expression of ZEB2 was significantly associated with lower survival rate of GBM patients (P=0.001). ZEB2, lower KPS score (P=0.004), gross total resection (P<0.001) and higher Ki67 percentage (P=0.001) were notably correlated to worse prognosis of GBM. With multivariate analysis, high expression of ZEB2 was demonstrated to be an independent prognostic factor indicating unfavorable prognosis of GBM (P=0.001, HR=3.86, and 95%CI=1.61–9.23).ConclusionsHigh expression of ZEB2 is an independent prognostic factor predicting unfavorable prognosis of GBM, indicating that ZEB2 or its downstream proteins may be potential drug targets of GBM therapy.

Role of C terminal binding proteins (CtBP) in pancreatic adenocarcinoma (PDAC).

320 Background: Overexpression of the oncogenic transcription factors CtBP1 and 2 is common and associated with poor prognosis in ovarian, breast and prostate cancer. CtBP2 drives tumor progression through promotion of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) activity. Frequent early metastasis observed in PDAC may depend on EMT and CSC activity, prompting us to investigate the prognostic significance of CtBP1/2 in PDAC. In parallel, we determined the oncogenic contribution of CtBP2 to PDAC utilizing a transgenic KRAS mutant mouse PDAC model (CKP) that incorporated heterozygous loss of the Ctbp2 gene (CKP2). Methods: Immunohistochemistry (IHC) using CtBP1/2 antibodies was performed on 69 cases of resected PDAC from VCU Health for the period 2005-16. Clinical data included stage (84% stage 1/2; 16% stage 3/4), initial CA19-9 level, and overall survival. IHC stains were assigned an Allred score (sum of intensity and proportion) by two independent pathologists with a scale from 0-8. Cox Proportional Hazard survival models were fit for CtBP1/2 staining vs. survival. CtBP1/2 correlation with stage and CA19-9 level was tested by ANOVA. CKP mice were bred to Ctbp2 +/- mice, and survival of cohorts (n = 10 each) of CKP vs. CKP2 mice was analyzed by log-rank analysis of Kaplan-Meier survival curves. Results: CtBP1 and 2 expression was uniformly high (7-8) across all cases, including stage 1/2. Thus, no statistically significant survival difference could be derived based on CtBP1/2 expression [CtBP1 HR 1.03 (0.67-1.41) p 0.92, CtBP2 HR 0.75 (0.39-1.43) p 0.31], and no significant association of CtBP1/2 was seen with stage or CA19-9. Suggesting a possible contribution of CtBP2 in human PDAC, Ctbp2 knockout in CKP2 mice improved median survival (9.5 vs. 8.1 weeks, p &lt; 0.05), decreased mean tumor weight (750 mg vs. 400 mg, p &lt; 0.05), and also abrogated peritoneal metastases and ascites normally seen in 100% of CKP mice. Conclusions: While uniformly high CtBP expression in PDAC subverts prognostic use, CtBP may play a pathogenic role, as CKP2 mice exhibited improved survival, lower tumor burden and no peritoneal spread. Our data supports further study of CtBP inhibition as a potential therapeutic strategy in PDAC.

The A2B Adenosine Receptor Modulates the Epithelial- Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells.

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies.

GP73 promotes epithelial-mesenchymal transition and invasion partly by activating TGF-β1/Smad2 signaling in hepatocellular carcinoma.

The transforming growth factor-β1 (TGF-β1) signaling pathways contribute to cell metastasis and epithelial-mesenchymal transition (EMT). Golgi protein 73 (GP73), a type II transmembrane protein in the Golgi apparatus, was initially regarded as a potential biomarker for the diagnosis of hepatocellular carcinoma (HCC). Recently, it was reported that GP73 acts as a key oncogene by promoting HCC growth and metastasis. However, the role of GP73 in metastasis, especially when involving signaling pathways, is uncertain. Here, we report that GP73, which is upregulated in HCC tissues and cell lines, is associated with tumor size, tumor node metastasis stage, distant metastasis and vascular invasion. The ectopic overexpression of GP73 increased HCC cell invasion, EMT and metastasis both in vitro and in vivo. Conversely, GP73 knockdown inhibited invasion and EMT. Moreover, GP73 enhanced p-Smad2 and p-Smad3 levels by mediating TGF-β1, thus leading to the promotion of EMT and invasion in HCC cells. In contrast, we used SB431542 (SB) to repress p-Smad2 and p-Smad3 expression, which resulted in a reversion of EMT. Furthermore, when the TGF-β1/Smad pathway was blocked, upregulation of GP73 still caused an enhanced EMT and invasion, and downregulation of GP73 resulted in a decreased in EMT and invasion. In clinical HCC samples, GP73 positively correlated with TGF-β1/Smad2, which was upregulated in HCC. Taken together, our findings highlight the important role of GP73 in regulating EMT and metastasis in HCC partly by targeting TGF-β1/Smad2 signaling, suggesting that GP73 may represent a novel potential therapeutic target and prognostic marker for the treatment and diagnosis of HCC.

Overexpression of FGFR1 Promotes Peritoneal Dissemination Via Epithelial-to-Mesenchymal Transition in Gastric Cancer.

Peritoneal dissemination (PD) is one of the most common causes of cancer-related mortality in gastric cancer (GC). We aimed to identify PD-associated genes and investigate their role in GC. We identified FGFR1 as a putative PD-associated gene using a bioinformatics approach. The biological significance of FGFR1 in epithelial-to-mesenchymal transition (EMT) was evaluated according to the correlation with genes that participated in EMT and FGFR1 knockdown experiments. The associations between FGFR1 expression and the clinicopathological features were examined. FGFR1 expression positively correlated with SNAI1, VIM and ZEB1 expression, and negatively correlated with CDH1 expression. Knockdown of FGFR1 suppressed the malignant phenotype of GC cells. High FGFR1 expression significantly correlated with the peritoneal lavage cytology and synchronous PD positivity as well as poor prognosis. High FGFR1 expression was associated with PD via promotion of EMT and led to a poor prognosis of GC patients.

Abstract A070: Uncovering unknown Aurora kinase signaling in adrenal tumor cell line, NCI-H295

Abstract Adrenocortical carcinomas (ACC) are rare tumors with no effective therapy for patients with advanced or metastatic disease. Therefore, efforts have been made to develop new therapeutic approaches for effective ACC treatment. Studies suggest that the dysregulation of Aurora kinases might be strongly associated with tumorigenesis and that Aurora kinases are involved in the promotion of the epithelial-mesenchymal transition (EMT) and invasion in different cancer cells. Molecular markers for EMT include decreased expression of E-cadherin, nuclear localization of β-catenin, and upregulation of genes related to the Wnt/β-catenin pathway, such as Cyclin D1 and c-Myc. Thus, the aim of this study was to investigate whether Aurora kinases are involved in the promotion of EMT in ACC cells. NCI-H295 cells were treated with 50 nM of the pan-Aurora kinase inhibitor, AMG 900, for 6, 24, and 48 hours. Multiple Reaction Monitoring proteomic approach was performed to screen proteins that are known to be involved in EMT. Total and enriched membrane, nucleus, and cytosol proteins were obtained by different centrifugation grades and detergents. Western blotting was performed for protein expression and subcellular localization. Cell cycle, proliferation, and migration assays were performed to study the effect of AMG 900 in NCI-H295 cells. MRM studies showed increased expression of E-cadherin 24 and 48 hours after AMG 900 treatment and decreased expression of N-cadherin after 48 hours of treatment. Conversely, our data showed increased of β-catenin expression and Vimentin expression after 48 hours of treatment. Moreover, our results showed higher expression of β-catenin in the nucleus when compared to control. Western blotting analysis confirmed these findings and gene expression analysis showed increased of c-Myc, one of the main products of β-catenin nuclear translocation, after 24 hours of treatment. We performed functional assays in order to check whether the studied pathways would affect NCI-H295 cell proliferation and migration, and our results showed that inhibition of Aurora kinases leads to cell cycle arrest in mitosis and decreases cell proliferation and migration. Further studies need to be performed combining Aurora kinases and β-catenin inhibitors; however, our results suggest that Aurora kinases might regulate proteins involved in EMT process during ACC progression. Citation Format: Andrea G. Maria, Carolina H. Thomé, Kleiton S. Borges, Mariana L. Grassi, Augusto F. Andrade, Regia C.P. Lira, Fabio R. Faucz, Constantine A. Stratakis, Luiz G. Tone, Vitor M. Faca. Uncovering unknown Aurora kinase signaling in adrenal tumor cell line, NCI-H295 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A070.

Abstract A205: Q701, a selective Axl and Mer inhibitor as an immune checkpoint inhibitor

Abstract Axl and Mer receptors are reported to have many functions, including promotion of epithelial-mesenchymal transition (EMT), resistance to antitumor therap,y and negative regulation of the innate immune response. Particular attention has been given to Axl and Mer receptor tyrosine kinase's (RTK) roles in regulating macrophage and NK cell activation as tumor cells exploit Axl and Mer signaling pathway to downregulate antitumor immunity, constituting an immune checkpoint. Q701 is a highly potent, selective, orally available Axl and Mer RTK inhibitor that can relieve immune suppression through innate immunity activation leading to T-cell activation. In various syngeneic mouse models, Q701 shows tumor regression activities by treatment of compound alone or in combination with other treatments, while Q701 has no direct cytotoxic activity against tested cell lines. Q701 treatment also show immune activation profile in tumor leading to T-cell activation. Q701 is under IND-enabling studies and a back-up program with Axl, Mer, and CSF1R inhibitors is in candidate nomination stage. Citation Format: Yeong-In Yang, Hwankyu Kang, Dongsik Park, Jiye Ahn, Jinho Choi, Seohyun Ahn, Jaeseung Kim, Kiyean Nam. Q701, a selective Axl and Mer inhibitor as an immune checkpoint inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A205.

Characterization of circulating tumor cells in newly diagnosed breast cancer.

Identification of circulating tumor cells (CTCs) by surface marker expression and ploidy analysis [immunostaining-fluorescence in situ hybridization (iFISH)] has been shown to be a highly sensitive method in the identification of certain solid cancers. In the present study, iFISH analysis was performed to identify CTCs in 184 patients with newly diagnosed non-metastatic breast cancer, and the distribution of CTC subtypes was characterized based on cytokeratin (CK) expression and ploidy status. It was revealed that CTCs of non-metastatic, aneuploid breast cancers, independent of CK expression profile, can be detected with high sensitivity (90.76%) by the iFISH system. Higher CTC counts and sensitivity were observed in patients with increased tumor size burden and unfavorable hormone receptor status. Investigation of CTC subtypes based on ploidy analysis indicated that triploid CTCs constituted the majority of CTCs evaluated. While CK-positive CTCs were detected in a small cohort of patients, an overall low rate of CK expression was observed in the 18 patient samples evaluated. Of note, CK expression was rare in CTCs detected in patients with Herceptin 2 (Her2)-positive or triple-negative [estrogen receptor (ER)-, progesterone receptor (PR)- and Her2-negative], indicating that lack of ER and PR may result in promotion of epithelial-mesenchymal transition and enhancement of tumor aggression.

MiR-19a and miR-424 target TGFBR3 to promote epithelial-to-mesenchymal transition and migration of tongue squamous cell carcinoma cells

ABSTRACTPrevious studies indicate that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), a novel suppressor of progression in certain cancers, is down-regulated in tongue squamous cell carcinoma (TSCC). However, the role of this factor as an upstream regulator in TSCC cells remains to be elucidated. The present study was designed to elucidate whether TGFBR3 gene expression is regulated by two microRNA molecules, miR-19a and miR-424. The study also aimed to determine if these microRNAs promote migration of CAL-27 human oral squamous cells. Immunohistochemistry (IHC) and western blot analyses demonstrated that TGFBR3 protein levels were dramatically down-regulated in clinical TSCC specimens. Conversely, bioinformatics analyses and qRT-PCR results confirmed that both miR-19a and miR-424 were markedly up-regulated in clinical TSCC specimens. In this study, we observed that transfection of a TGFBR3-containing plasmid dramatically inhibited epithelial-to-mesenchymal transition (EMT) and migration in CAL-27 cells. Co-immunoprecipitation analyses also revealed that TGFBR3 forms a complex with the β-arrestin 2 scaffolding protein and IκBα. Furthermore, overexpression of TGFBR3 decreased p-p65 expression and increased IκBα expression; these effects were subsequently abolished following knockdown of β-arrestin 2. Moreover, over-expression of miR-19a and miR-424 promoted migration and EMT in CAL-27 cells. We also observed that the promotion of EMT by miR-19a and miR-424 was mediated by the inhibition of TGFBR3. Our study provides evidence that miR-19a and miR-424 play important roles in the development of TSCC. These results expand our understanding of TGFBR3 gene expression and regulatory mechanisms pertaining to miRNAs.