High Vimentin Research Articles

Abstract 1719: Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels

Abstract Introduction: Liquid biopsy based on sub-micron or nanosized particles in human body fluid have been received vast attention due to their non-invasive characteristics and enabling multiple check-up. Circulating tumor cells (CTCs), as well as exosome are the most promising markers in liquid biopsy, however, dual isolation and profiling have been hampered due to their size difference and limited quantity for analysis. We proposed the novel and simple methods for both isolation and study their similarity between them. Using the label-free CTC filtration device and anti-CD63 antibody-conjugated degradable hydrogel, the CTCs and the CTCs-derived exosome are specifically isolated, and each samples were followed by molecular study after recovery. This versatile platform facilitates the comprehensive study of two biomarkers with reflecting their inherent characteristics, thus paving the way for revealing their roles in cancer progression and metastasis. Methods: In order to make stimuli-responsive degradable hydrogel, poly (vinyl alcohol) and alginate were mixed under constant stirring at 85 °C. The mixture was poured into the mold and dried for 24 hours. Then, the dried sheet was immersed into 100 mM calcium chloride solution to achieve gelation through ionic interaction. Subsequently, the anti-CD63 antibody was immobilized onto the prepared hydrogels via cross-linking. For the dual-profiling, the hydrogel and the filters containing the captured breast cancer cells by microfiltration were incubated with the exosome-depleted cell culture media for 6 hours. The captured cells were released from the device and the hydrogels were degraded by adding EDTA. The cell and exosome lysate were prepared using RIPA buffer at 4 °C. The supernatant was collected by centrifugation followed by western-blot assay. Four different markers, including exosome-specific marker (CD63), cancer-associated markers (EpCAM, vimentin), and a housekeeping marker (β-actin), were used. Results: All exosome and cell samples highly expressed the housekeeping marker. Especially, two exosome samples dissociated from the hydrogel showed CD63 predominantly, which support the secretion of exosome from the cancer cells. The samples from the released cancer cells from the device did not express CD63 remarkably. To verify the phenotypical similarity between cell and exosome, expressions of the epithelial marker (EpCAM) and mesenchymal marker (vimentin) were examined. The exosome and cell from MCF-7, epithelial cancer cell, showed higher expression of EpCAM then vimentin. On the contrary to this, the samples from MDA-MB-231, mesenchymal cell, showed higher vimentin expression then EpCAM. Discussion and conclusion: We showed that exosome follow the phenotypical characteristics of mother-cells. This dual profiling would be helpful for in-depth study of cancer with consideration of its heterogeneity and complexity. Citation Format: Yoon-Tae Kang, Young Jun Kim, Tae Hee Lee, Jae-Eul Shim, Young-Ho Cho. Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1719. doi:10.1158/1538-7445.AM2017-1719

Stromal Expression of Vimentin Predicts the Clinical Outcome of Stage II Colorectal Cancer for High-Risk Patients.

BackgroundIncreased expression of vimentin in tissue samples from patients with colorectal cancer (CRC) has been previously demonstrated, but its prognostic significance remains controversial, and the clinical significance for patients with stage II CRC is still unknown. The aim of this study was to evaluate the expression of vimentin in CRC and its potential prognostic significance.Material/MethodsWe analyzed vimentin expression in 203 CRC tissue samples from patients with stage II cancer using immunohistochemistry, and correlated the findings with clinicopathological patient features. CRC-specific survival (CSS) and disease-free survival (DFS) were analyzed using the Kaplan-Meier method. Univariate and multivariate analysis was performed using the Cox proportional hazards method for survival.ResultsVimentin expression was significantly correlated only with tumor (T) stage (p=0.024). Kaplan-Meier survival analysis indicated that vimentin expression could stratify the CSS and DFS of patients with stage II CRC at high risk (p=0.029, p=0.042, respectively), but not those of low-risk stage II patients (p=0.208, p=0.361, respectively). Univariate and multivariate analysis further revealed that stromal vimentin expression is an independent prognostic factor for CSS and DFS of high-risk stage II patients (p=0.043, p=0.022, respectively). Moreover, high-risk stage II patients with low stromal vimentin expression benefitted more from standard adjuvant chemotherapy than those with high stromal vimentin expression (CSS: p=0.012 vs. p=0.407; DFS: p=0.017 vs. p=0.420).ConclusionsOur study suggests that stromal vimentin expression is a promising indicator for survival prediction and adjuvant chemotherapy response in patients with stage II CRC with high-risk factors for recurrence.

Vimentin is a potential prognostic factor for tongue squamous cell carcinoma among five epithelial-mesenchymal transition-related proteins.

We aimed to investigate the association of the expression levels of five epithelial–mesenchymal transition (EMT)-related proteins (Snail, Twist, E-cadherin, N-cadherin, and Vimentin) with tumorigenesis, pathologic parameters and prognosis in tongue squamous cell carcinoma (TSCC) patients by immunohistochemistry of tissue microarray. The expression levels of Snail, E-cadherin, N-cadherin and Vimentin were significantly different between the tumor adjacent normal and tumor tissues. In tumor tissues, lower E-cadherin and higher N-cadherin levels were associated with a higher grade of cell differentiation, advanced stage of disease, and lymph node metastasis. However, higher Vimentin expression was associated with poor cell differentiation and lymph node metastasis. Patients with low E-cadherin expression had poor disease-specific survival (DSS). Conversely, positive N-cadherin and higher Vimentin expression levels were associated with poor DSS and disease-free survival. Notably, our multivariate Cox regression model indicated that high Vimentin expression was an adverse prognostic factor for DSS in TSCC patients, even after the adjustment for cell differentiation, pathological stage, and expression levels of Snail, Twist, E-cadherin, and N-cadherin. Snail, E-cadherin, N-cadherin, and Vimentin were associated with tumorigenesis and pathological outcomes. Among the five EMT-related proteins, Vimentin was a potential prognostic factor for TSCC patients.

Abstract TMEM-038: THE CULTIVATION AND PARTITIONING OF INVASIVE OVARIAN CANCER CELLS AFTER THEIR ISOLATION FROM WHOLE BLOOD

Abstract Ovarian cancer patients suffer from high rates of recurrence and mortality. Circulating tumor cells (CTCs) can serve as a minimally invasive tool to improve the clinical management of such cancer patients. Hence there is growing interest in the isolation, characterization and in vitro culture of CTCs. To this end, the porous membrane cell separators developed by MetaCell s.r.o. provide a simple solution. Ovarian cancer cell line models OVCAR3 and SKOV3 were studied by gene expression profiling and immunostaining for the expression of EPCAM, EGFR, HER2, MET and certain other novel therapeutic targets. Cultivation of the cancer cells after their spike recovery from either cell culture medium or from whole blood was explored using MetaCellTM separators. The cells growing on the separator membranes housed in multi-well culture plates was conveniently visualized with vital cell stains. The SKOV3 cells, but not the OVCAR3 cells, were found to migrate through the membrane pores and populate the well bottoms of the culture plates. This can be attributed to the more plastic and invasive nature of SKOV3 cells as characterized by high Vimentin and low EPCAM expression levels. This is akin to the profile exhibited by highly invasive cancer cells that have undergone epithelial to mesenchymal transition (EMT) and that are implicated in the metastatic spread of cancer in patients. Furthermore, the invasive cells growing on the culture plate well bottoms were found to still stain positively for the therapeutic targets of interest. This cultivation device thus offers the ability to selectively interrogate the more invasive sub-populations within the highly heterogeneous CTCs that are typically seen in cancer patients. After the isolation and cultivation of CTCs, if the partitioned invasive cells remain addressable for the therapeutic target under consideration, the patients are more likely to respond to that therapy for the arrest of metastatic disease. In this context, the MetaCellTM separators have the potential to serve as very effective adjunctive aids for the selection and monitoring of ovarian cancer patients for treatment with targeted therapies. Citation Format: Uday K. Veeramallu, M.S. THE CULTIVATION AND PARTITIONING OF INVASIVE OVARIAN CANCER CELLS AFTER THEIR ISOLATION FROM WHOLE BLOOD [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-038.

MiR-551b regulates epithelial-mesenchymal transition and metastasis of gastric cancer by inhibiting ERBB4 expression.

Epithelial-mesenchymal transition (EMT) is an important biological process that is characteristic of malignant tumor cells with metastatic potential. We investigated the role of miR-551b in EMT and metastasis in gastric cancer (GC). We found that low miR-551b levels were associated with EMT, metastasis and a poor prognosis in GC patients. Further, two GC cell lines, MNK45 and SGC7901, exhibited lower miR-551b levels than the GES normal stomach cell line. Exposing MNK45 and SGC7901 cells to TGF-β1 resulted in cell morphology changes characteristic of EMT, which was confirmed by Western blot analysis demonstrating low E-Cadherin and high N-Cadherin and Vimentin levels. Treatment with miR-551b mimics inhibited these EMT changes as well as Transwell migration and invasiveness. We identified ERBB4 as a potential target of miR-551b based on patient data from the TCGA. ERBB4 was upregulated in GC specimens, and its high expression correlated with a poor prognosis of GC patients. Dual luciferase assays revealed that miR-551b directly inhibited ERBB4 by binding to its 3′UTR. Moreover, treatment with miR-551b mimics or the ERBB4 inhibitor AST-1306 inhibited EMT in the GC cell lines. Finally, nude mice xenografted with GC cancer cell lines expressing miR-551b mimics exhibited smaller tumors and longer survival than mice engrafted with control GC cancer cells. These data indicate that miR-551b inhibits EMT and metastasis in GC by inhibiting ERBB4. miR-551b and ERBB4 are thus potential therapeutic targets for the treatment of GC.

Epithelial-Mesenchymal Transition in Non Small-cell Lung Cancer.

Lung cancer is the first cause of cancer related deaths in both males and females. Epithelial-mesenchymal transition (EMT) is a reversible process by which epithelial cells transform to mesenchymal stem cells by losing their cell polarity and cell-to-cell adhesion, gaining migratory and invasive properties. High levels of E-cadherin are expressed in epithelial cells, whereas mesenchymal cells express high levels of N-cadherin, fibronectin and vimentin. The aim of this study was to evaluate the correlation between E-cadherin and vimentin expression and their clinical significance in non-small cell lung cancer (NSCLC). The immunohistochemical expression of E-cadherin, vimentin and Ki-67 was performed on tissue microarrays from NSCLC specimens obtained from 112 newly- diagnosed cases and were studied using classical pathological evaluation. Associations between E-cadherin, vimentin and Ki-67 expression, clinicopathological variables and survival were analyzed. In all cases, a value of p≤0.05 was considered significant. Low E-cadherin expression was significantly correlated with tumor necrosis (p=0.019). Moreover, there was a trend for correlation between high E-cadherin expression and better overall survival (hazard ratio=1.02, and 95% confidence interval=0.45-1.87, p=0.091). There was also a significant negative correlation between vimentin expression and overall survival (hazard ratio=1.13, and 95% confidence interval=0.78-1.65, p=0.026). Additionally, there was a significant negative correlation between vimentin expression and grade I tumors (p=0.031). Finally, a positive correlation trend between vimentin expression and Ki-67 was found (p=0.073). High E-cadherin and low vimentin expression correlate with better prognosis and overall survival.

Expression of epithelial and mesenchymal markers in invasive (ductal) breast carcinoma and nodal metastasis

Introduction The phenomenon, Epithelial-mesenchymal transition (EMT) has been shown to enable epithelial cells to acquire a mesenchymal phenotype facilitating cancer cell mobilization. Evidence in favour has been derived predominantly from cancer cell lines and/or animal model studies. The objectives of this study were to observe E-cadherin and vimentin expression patterns in invasive (ductal) breast carcinoma-no special type (BCa) and corresponding metastatic lymph nodes (mLNs) and to compare the expression of these markers in BCa with and without metastasis using clinical samples. Methodology Tissues of 24 metastatic BCa (mBCa) with mLNs and 17 non metastatic BCa (nmBCa) obtained from the Department of Pathology, Faculty of Medicine, Peradeniya were included. Immunohistochemical expression of E-cadherin (epithelial cell adhesion membrane marker) and vimentin (mesenchymal cytoplasmic marker) was observed on all BCas and mLNs. Ten random high-power fields (10x40) of 100 cells each were assessed to determine the percentage of positive cells . The mean expression of markers was compared between mBCa and nmBCa by independent sample T-test and mBCa and mLN by paired sample T-test. Results The mean E-cadherin expression was 70.54% (27.67%-97.67%) in mBCa, 86.40% (63.33%-96.67%) in nmBCa and 89.95% (46.33%-100%) in mLNs. The mean vimentin expression was 6.54% (2.37%-13.87%) in mBCa, 5.39% (2.12%- 9.77%) in nmBCa and 1.28% (0.13%-8.25%) in mLNs. The E-cadherin expression was significantly lower in mBCa compared to nmBCa (p=0.002) and in mBCa compared to mLNs (p=0.000). Vimentin expression was not significant between mBCA and nmBCa (p=0.176). Higher vimentin expression in mBCa compared to mLNs was statistically significant (p=0.000). Discussion and conclusion The significantly lower expression of E-cadherin in mBCa compared to nmBCa suggests that loss of epithelial cell properties promotes metastasis. Significantly lower E-cadherin and significantly higher vimentin expressions in mBCa compared to mLNs is supportive of EMT in BCa. The reversed pattern is noted in nodal BCa metastasis suggesting the possibility of mesenchymal-epithelial transition of metastatic cancer cells in the lymph node.

Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer Patients.

Extensive stromal interaction is one reason for the dismal outcome of biliary tract cancer (BTC) patients. Epithelial to mesenchymal transition (EMT) is involved in tumor invasion and metastasis and is partly regulated by microRNAs (miRs). This study explores the expression of anti-EMT miR200 family (miR141, −200a/b/c, −429) and miR205 as well as the EMT-related proteins E-cadherin and vimentin in a panel of BTC cell lines and clinical specimens by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry, respectively. MicroRNA expression was correlated to (i) the expression patterns of E-cadherin and vimentin; (ii) clinicopathological characteristics; and (iii) survival data. MicroRNA-200 family and miR205 were expressed in all BTC cells and clinical specimens. E-cadherin and vimentin showed a mutually exclusive expression pattern in both, in vitro and in vivo. Expression of miR200 family members positively correlated with E-cadherin and negatively with vimentin expression in BTC cells and specimens. High expression of miR200 family members (but not miR205) and E-cadherin was associated with longer survival, while low miR200 family and high vimentin expression was a predictor of unfavorable survival. Overall, the current study demonstrates the relevance of the miR200 family in EMT of BTC tumors and suggests these miRs as predictors for positive outcome.

Relationship and clinicopathological significance of Numb and epithelial-mesenchymal transition related proteins in human pancreatic cancer

Objective: To study the relationship and clinicopathological significance of Numb and epithelial-mesenchymal transition related proteins in human pancreatic cancer(PC). Methods: Sixty-three cases of pancreatic cancer tissues were obtained from department of gastrointestinal surgery in the First Hospital of China Medical University from January 2005 to December 2012, all samples were histopathologically proved to be adenocarcinoma. The expressions of Numb, E-cadherin and Vimentin proteins in 63 cases of pancreatic cancer specimens were detected by immunohistochemistry. Western blot and real-time PCR were used to examine the protein and mRNA levels in two pancreatic cancer cell lines. Pearson and chi-squared tests were used to analyze the relationship and clinicopathological characters with PC patients. Kaplan-Meier curve and log rank test were used to estimate the difference of PC patients' survival. Results: The positive rates of Numb, E-cadherin and Vimentin expressions were 46.0%, 41.3% and 28.6%, respectively. Numb expression was negatively associated with tumor size, differentiation and UICC stage(r=-0.310, P=0.010; r=-0.359, P=0.004; r=-0.228, P=0.020), while E-cadherin expression was negatively related with tumor differentiation(r=-0.316, P=0.012). In contrast, Vimentin expression was positively related with pancreatic cancer differentiation and lymph metastasis(r=0.264, P=0.036; r=0.274, P=0.030). Correlation analysis showed Numb had a positive association with E-cad expression(r=0.325, P=0.010), but had no association with Vimentin. Moreover, patients with co-expression of Numb and E-cadherin had a significantly better overall survival in Kaplan-Meier univariate analysis(P=0.046). Immunoblotting and real-time PCR showed that high Numb protein and mRNA levels in BxPC-3 cells were followed with high E-cadherin and low Vimentin expressions, whereas low Numb protein and mRNA levels in PANC-1 cells were followed with low E-cadherin and high Vimentin expressions, respectively. Conclusions: Numb has a positive relationship with E-cadherin in both pancreatic cancer tissues and cells.The interaction between them might participate in the initiation and development of epithelial-mesenchymal transition in pancreatic cancer.

High Vimentin Expression Associated with Lymph Node Metastasis and Predicated a Poor Prognosis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a common public health problem worldwide with poor prognosis, which is largely due to lymph node metastasis and recurrence. Identification of specific molecular markers of OSCC with lymph node metastasis would be very important for early and specific diagnosis. In this study, we screened for the potential prognosis markers via unbiased transcriptomic microarray analysis in paired two OSCC cell lines, a lymph node metastatic HN12 cell line and a low metastatic parental HN4 cell line. The results showed that vimentin, with 87-fold increase of expression, was on the top of all upregulated genes in metastatic HN12 cells compared to non-metastatic HN4 cells. Treatment of non-metastatic HN4 cells with TGF-β1 induced epithelial to mesenchymal transition (EMT), with increased vimentin expression as well as enhanced migration activity. Consistently, knockdown of vimentin via siRNA resulted in suppressed invasion and migration activities of HN12 cells, suggesting an essential role of vimentin in EMT-related functions of OSCC cells. Finally, immunohistochemical (IHC) staining analysis showed that high vimentin expression was strongly associated with high lymph node metastases (p < 0.05), and poor overall survival (p < 0.05) in OSCC patients. Thus, high vimentin expression is strongly associated with increased metastatic potential, and may serve as a prediction marker for poor prognosis in OSCC patients.

Abstract A113: The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with the worst survival rate among solid cancers. The pressing needs for extending life expectancy of patients are the identification of early prognostic markers and novel druggable pathways. PDAC arises generally from pancreatic intraepithelial neoplasia (PanIN) and a dynamic interactions between tumor, stromal cells and autocrine and paracrine signaling lead to epithelial to mesenchymal transition (EMT), an early process in the natural history of pancreatic cancer. Cytoskeletal reorganization, extracellular matrix (ECM) remodeling, and matrix metalloproteinases (MMPs) contribute to PDAC aggressiveness in cooperation with soluble growth factors or cytokines, with TGF-β1 as crucial player. hMENA is an actin regulatory protein whose splicing program, mediated by the epithelial splicing regulatory proteins (ESRPs), has been associated with the EMT process. Our previous studies indicated that alternative splicing of hMENA, generates hMENA11a and hMENAΔv6 isoforms with opposite roles in cell proliferation and invasion in breast and lung cancers. Alternative splicing is known to play a prominent role in tumor progression and tumorigenesis and the derived isoforms may represent powerful diagnostic and prognostic factors as we have recently shown for hMENA alternative splicing in early stage non-small cell lung cancer (NSCLC). The aim of this study is to investigate the role of TGF-β1 on the expression and function of hMENA isoforms in PDAC, and verify whether the expression pattern of hMENA isoforms may impact patient outcome. Methods: We analyzed the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas, evaluating the patient outcome. The functional role of hMENA isoforms were analyzed by loss and gain of function experiments in untreated and TGF-β1-treated PDAC cell lines. Results: In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, while hMENAΔv6 expression with a mesechymal phenotype, with low E-cadherin and high vimentin expression. hMENA11a knock-down in PDAC cell lines affected cell-cell adhesion but not cell invasion. TGF-β1 cooperated with β-catenin signalling to up-regulate hMENA and hMENAΔv6 expression but not hMENA11a. The hMENA/hMENAΔv6 up-regulation play a crucial role in cell invasiveness and in TGF-β1-induced EMT. After TGF-β1 treatment, hMENA/hMENAΔv6 were mobilized from focal adhesion to actin stress fibers, and the silencing of these isoforms significantly inhibited the TGF-β1-induced EMT in PANC-1. Functionally, in the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulationis crucial for SMAD2-mediated TGF-β1 signalling, migration, invasion and MMPs activities. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome, in agreement with experimental results. Conclusions: hMENA isoforms are regulated differently by TGF-β1, and the pattern of expression of hMENA isoforms is crucial in TGF-β1-dependent EMT and cell invasion. The pattern of expression of hMENA isoforms correlates with PDAC patient outcome and it could be used in specific clinical settings for the choice of the most effective treatment of PDAC patients. Our data provide new insights into molecular pathways involved in PDAC biology and suggest that hMENA-related pathways are promising targets for the development of new prognostic and therapeutic tools in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Isabella Sperduti, Matteo Fassan, Ivana Cataldo, Borislav C. Rusev, Rita T. Lawlor, Maria Grazia Diodoro, Michele Milella, Gian Luca Grazi, Mina J. Bissell, Aldo Scarpa, Paola Nisticò. The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A113.

Abstract 1602: Snail transcription factor regulates nuclear cathepsin L activity

Abstract Triple negative breast cancer (TNBC) are defined as tumors lacking the expression of estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor-2, which accounts for approximately 15% of total breast cancer patients, and is more prevalent and lethal among young African, African-American and Latino women patients. The primary cause of breast cancer death is metastasis, which is regulated by factors such as epithelial mesenchymal transition (EMT), a dynamic process that promotes cell motility and decreases cell-cell adhesion. Snail transcription factor promotes EMT by repressing epithelial genes such as E-cadherin, while increasing mesenchymal genes such as vimentin. Cathepsin L (Cat L) cysteine protease promotes cell invasion and nuclear Cat L has recently been associated with poor prognosis in colon and breast cancer. However, the mechanism by which Cat L localizes to the nucleus is unknown. CDP/CUX transcription factor is a substrate for Cat L which when proteolytically cleaved by Cat L from the p200 to the p110 isoform can transcriptionally activate Snail and repress E-cadherin. We have recently published that Snail overexpression increases Cat L activity in prostate cancer cells which can be antagonized by muscadine grape skin extract (MSKE), a natural product rich in anthocyanin. We hypothesize that a feedback loop exists whereby Snail Transcription factor may promote nuclear Cat L expression and activity in TNBC which subsequently cleaves CDP/CUX and further increases Snail transcription, leading to increased EMT; this can be abrogated by Cat L inhibitor or MSKE. We utilized various breast cancer cell lines as well as MCF-7 cells stably overexpressing Snail (MCF-7 Snail) or control (MCF-7 Neo) to evaluate the expression of Snail, Cat L, CDP/CUX and EMT marker expression (E-cadherin, vimentin) by western blot and immunofluorescence. Migration and invasion assays were performed including treatments with Cat L specific inhibitor (Z-FY-CHO) or MSKE. We observed more Snail, Cat L and CDP/CUX cleavage products (p110 and p90 isoforms) expression in MCF-7 Snail cells and TNBC cells (MDA-MB-231, MDA-MB-468) as compared to MCF-7 control cells. Interestingly, immunofluorescent and cell fractionation analyses revealed that Snail overexpression promoted nuclear Cat L and CDP/CUX p110 isoform expression, and EMT (low E-cadherin, high vimentin, migration and invasion) which could be abrogated by Z-FY-CHO or MSKE. We are currently staining for Snail and Cat L in breast cancer tissue of varying races/grades including TNBC, as well as confirming by chromatin immunoprecipitation that the CDP/CUX cleavage products seen in TNBC can bind Snail and/or E-cadherin promoters. In conclusion, our study shows that Snail promotes nuclear localization of Cat L which may promote EMT via CDP/CUX, and that inhibition with Cat L inhibitors or natural products such as MSKE may be a good therapeutic target for TNBC. Citation Format: Liza J. Burton, Valerie Odero-Marah. Snail transcription factor regulates nuclear cathepsin L activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1602.

LPS/TLR4 Signaling Enhances TGF-β Response Through Downregulating BAMBI During Prostatic Hyperplasia.

Compelling evidence suggests that benign prostatic hyperplasia (BPH) development involves accumulation of mesenchymal-like cells derived from the prostatic epithelium by epithelial-mesenchymal transition (EMT). Transforming growth factor (TGF)-β induces EMT phenotypes with low E-cadherin and high vimentin expression in prostatic epithelial cells. Here we report that LPS/TLR4 signalling induces down-regulation of the bone morphogenic protein and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signalling in the EMT process during prostatic hyperplasia. Additionally, we found that the mean TLR4 staining score was significantly higher in BPH tissues with inflammation compared with BPH tissues without inflammation (5.13 ± 1.21 and 2.96 ± 0.73, respectively; P < 0.001). Moreover, patients with inflammatory infiltrate were more likely to have a higher age (P = 0.020), BMI (P = 0.026), prostate volume (P = 0.024), total IPSS score (P = 0.009) and IPSS-S (P < 0.001). Pearson’s correlation coefficient and multiple regression analyses demonstrated that TLR4 mRNA expression level was significantly positively associated with age, BMI, serum PSA levels, urgency and nocturia subscores of IPSS in the inflammatory group. These findings provide new insights into the TLR4-amplified EMT process and the association between TLR4 levels and storage LUTS, suggesting chronic inflammation as vital to the pathogenesis of BPH.

A new patient-derived orthotopic malignant meningioma model treated with oncolytic herpes simplex virus.

Higher-grade meningiomas (HGMs; World Health Organization grades II and III) pose a clinical problem due to high recurrence rates and the absence of effective therapy. Preclinical development of novel therapeutics requires a disease model that recapitulates the genotype and phenotype of patient HGM. Oncolytic herpes simplex virus (oHSV) has shown efficacy and safety in cancers in preclinical and clinical studies, but its utility for HGM has not been well characterized. Tumorsphere cultures and serial orthotopic xenografting in immunodeficient mice were used to establish a patient-derived HGM model. The model was pathologically and molecularly characterized by immunohistochemistry, western blot, and genomic DNA sequencing and compared with the patient tumor. Anti-HGM effects of oHSV G47Δ were assessed using cell viability and virus replication assays in vitro and animal survival analysis following intralesional injections of G47Δ. We established a serially transplantable orthotopic malignant meningioma model, MN3, which was lethal within 3 months after tumorsphere implantation. MN3 xenografts exhibited the pathological hallmarks of malignant meningioma such as high Ki67 and vimentin expression. Both the patient tumor and xenografts were negative for neurofibromin 2 (merlin) and had the identical NF2 mutation. Oncolytic HSV G47Δ efficiently spread and killed MN3 cells, as well as other patient-derived HGM lines in vitro. Treatment with G47Δ significantly extended the survival of mice bearing subdural MN3 tumors. We established a new patient-derived meningioma model that will enable the study of targeted therapeutic approaches for HGM. Based on these studies, it is reasonable to consider a clinical trial of G47Δ for HGM.

PD-L1 expression is associated with epithelial-mesenchymal transition in head and neck squamous cell carcinoma.

Virus-associated malignancies and sarcomatoid cancers correlate with high PD-L1 expression, however, underlying mechanisms remain controversial. We evaluated the correlation between PD-L1 expression and epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinomas (HNSCC). Tumor tissues from 50 patients with HNSCC were evaluated for PD-L1 by immunohistochemistry, which showed 32 (64.0%) were PD-L1 positive (PD-L1+). Interestingly, PD-L1 expression was significantly associated with EMT (P = 0.010), as assessed by low E-cadherin and high vimentin expression. The overall survival of PD-L1+ patients with EMT features was significantly worse than those without EMT features (P = 0.007). In an independent validation cohort (N = 91), as well as in HNSCC cases of The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia, high PD-L1 expression was also associated with the high probability of an EMT signature, referred from the GEO dataset, GSE4824. Survival analysis confirmed PD-L1+/EMT+ patients had a poorer prognosis than PD-L1+/EMT- patients in the TCGA cohort. PD-L1 positivity can thus be divided into two categories according to the absence or presence of EMT. PD-L1 expression is also independently associated with EMT features in HNSCC.

Abstract A70: Akt2 gain or Akt1 loss drives invasion and aggressiveness in breast cancer

Abstract Despite the improvement in the early diagnosis and the use of modern therapies, breast cancer is the second cause of death, after lung cancer, in women around the world, and this is mainly due to metastasis development distant organs. PI3K/Akt pathway is deregulated in almost 50% of breast carcinomas, and is associated to therapy resistance. Currently, there are increasing clinical trials that combine endocrine treatments with PI3K/Akt inhibitors to delay tumor resistance and to avoid tumor progression. However, these inhibitors do not distinguish between the isoform specific function of Akt1, Akt2 and Akt3. Several evidences in different models of cancer show that Akt1 and Akt2 are expressed in different stages of cancer progression, with Akt1 involved in tumor proliferation during the first steps of the carcinogenic process, and Akt2 involved in the invasive phenotype. The aim of this work was to study Akt1 and Akt2 isoforms, focusing on their particular role in cell migration and invasion in human breast tumor samples and in two human breast cancer cell lines. Tissue microarray analysis in a small cohort of human advanced breast cancer samples showed a positive association between invasive grade, Akt2 expression and E-cadherin loss. Interestingly, E-cadherin presents a heterogeneous staining in each sample, being weaker in the more invasive areas and stronger in the non-invasive areas. To understand the specific role of each isoform of Akt on tumor phenotype, T47D and IBH-6 human breast cancer cell lines were stably transfected to specifically inhibit Akt1 (shAkt1) or Akt2 (shAkt2) and the resulting phenotype in cell culture and in xenografts were analyzed. We found that Akt1 silencing inhibited cell growth through S6 and cyclin-D1 regulation, but induced cell migration and invasion in IBH-6 2D and 3D cell cultures. Contrarily, Akt2 silencing had no effect on cell proliferation but significantly inhibited cell adhesion and invasion. These phenotypes were accompanied by an opposite regulation of β1-Integrin, FAK, MMP9, F-actin and vimentin expression. Simultaneous silencing of Akt1 and Akt2 isoforms showed that Akt1 has an anti-migratory effect per se and not by upregulating Akt2 levels. Interestingly, in IBH-6 and T47D xenograft studies we observed that shAkt1 cells grew significantly more slowly than control tumors, but displayed a ducto-lobulillar invasive phenotype, similar to the phenotype gained with Akt2 overactivation by myristoylated Akt2. Contrarily, shAkt2 tumors showed no differences in growth rate, with no infiltrating morphology when compared to control tumors. Immunohistochemical analysis showed low E-cadherin and high vimentin expression in invasive T47D shAkt1 tumors, and the opposite pattern in T47D shAkt2 tumors. We are currently assessing Akt1 status, vimentin, β1-integrin, FAK and MMP9 levels in human samples to relate them with in vitro and xenografts results. Altogether, these results indicate that Akt2 overexpression as well as Akt1 inhibition play important roles in the acquisition of an invasive and aggressive phenotype. The status of each isoform-driven pathway is relevant in the design of targeted therapies that could be more effective and selective in each tumor stage. Citation Format: Cecilia Perrone, Marina Riggio, Maria May, Maria Laura Polo, Jimena Rodriguez, Diego Lucas Kaen, Claudia Lanari, Virginia Novaro. Akt2 gain or Akt1 loss drives invasion and aggressiveness in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A70.

The Superficial Stromal Scar Formation Mechanism in Keratoconus: A Study Using Laser Scanning In Vivo Confocal Microscopy.

To investigate the mechanism of superficial stromal scarring in advanced keratoconus using confocal microscopy, the keratocyte density, distribution, micromorphology of corneal stroma, and SNP in three groups were observed. Eight corneal buttons of advanced keratoconus were examined by immunohistochemistry. The keratocyte densities in the sub-Bowman's stroma, anterior stroma, and posterior stroma and the mean SNP density were significantly different among the three groups. In the mild-to-moderate keratoconus group, activated keratocyte nuclei and comparatively highly reflective ECM were seen in the sub-Bowman's stroma, while fibrotic structures with comparatively high reflection were visible in the anterior stroma in advanced keratoconus. The alternating dark and light bands in the anterior stroma of the mild-to-moderate keratoconus group showed great variability in width and direction. The wide bands were localized mostly in the posterior stroma that corresponded to the Vogt striae in keratoconus and involved the anterior stroma only in advanced keratoconus. Histopathologically, high immunogenicity of α-SMA, vimentin, and FAP was expressed in the region of superficial stromal scarring. In vivo confocal microscopy revealed microstructural changes in the keratoconic cone. The activation of superficial keratocytes and abnormal remodeling of ECM may both play a key role in the superficial stromal scar formation in advanced keratoconus.

NKX6.1 functions as a metastatic suppressor through epigenetic regulation of the epithelial-mesenchymal transition.

The transcription factor NKX6.1 (NK6 homeobox 1) is important in the development of pancreatic β-cells and neurons. Although recent publications show that NKX6.1 is hypermethylated and downregulated during tumorigenesis, the function of NKX6.1 in carcinogenesis remains elusive. Here, we address the metastasis suppressor function of human NKX6.1 using cell, animal and clinical analyses. Our data show that NKX6.1 represses tumor formation and metastatic ability both in vitro and in vivo. Mechanistically, NKX6.1 suppresses cell invasion by inhibiting the epithelial-to-mesenchymal transition (EMT). NKX6.1 directly enhances the mRNA level of E-cadherin by recruiting BAF155 coactivator and represses that of vimentin and N-cadherin by recruiting RBBP7 (retinoblastoma binding protein 7) corepressor. Clinical cancer tumors with metastasis show low NKX6.1 protein expression coinciding with low E-cadherin and high vimentin expression. Our results demonstrate that NKX6.1 functions as an EMT suppressor by interacting with different epigenetic modifiers, making it a potential novel therapeutic option.

MPGES-1 in prostate cancer controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity.

There is evidence that an inflammatory microenvironment is associated with the development and progression of prostate cancer (PCa), although the determinants of intrinsic inflammation in PCa cells are not completely understood. Here we investigated whether expression of intrinsic microsomal PGE synthase-1 (mPGES-1) enhanced aggressiveness of PCa cells and might be critical for epidermal growth factor receptor (EGFR)-mediated tumour progression. In PCa, overexpression of EGFR promotes metastatic invasion and correlates with a high Gleason score, while prostaglandin E2 (PGE2) has been reported to modulate oncogenic EGFR-driven oncogenicity. Immunohistochemical studies revealed that mPGES-1 in human prostate tissues is correlated with EGFR expression in advanced tumours. In DU145 and PC-3 cell lines expressing mPGES-1 (mPGES-1SC cells), we demonstrate that silencing or ‘knock down’ of mPGES-1 (mPGES-1KD) or pharmacological inhibition by MF63 strongly attenuates overall oncogenic drive. Indeed, mPGES-1SC cells express stem-cell-like features (high CD44, β1-integrin, Nanog and Oct4 and low CD24 and α6-integrin) as well as mesenchymal transition markers (high vimentin, high fibronectin, low E-cadherin). They also show increased capacity to survive irrespective of anchorage condition, and overexpress EGFR compared to mPGES-1KD cells. mPGES-1 expression correlates with increased in vivo tumour growth and metastasis. Although EGFR inhibition reduces mPGES-1SC and mPGES-1KD cell xenograft tumour growth, we show that mPGES-1/PGE2 signalling sensitizes tumour cells to EGFR inhibitors. We propose mPGES-1 as a possible new marker of tumour aggressiveness in PCa.

Absence of keratins 8 and 18 in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: Cell line selective effects on cell invasion

Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 mRNA levels being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription and mRNA or protein stability.