Low E-cadherin Expression Research Articles

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.

Ascochlorin Enhances the Sensitivity of Doxorubicin Leading to the Reversal of Epithelial-to-Mesenchymal Transition in Hepatocellular Carcinoma.

Increasing evidence has indicated that epithelial-to-mesenchymal transition (EMT) at the advanced stage of liver cancer not only has the ability to self-renew and progress cancer, but also enables greater resistance to conventional chemo- and radiotherapies. Here, we report that ascochlorin (ASC), an isoprenoid antibiotic, could potentiate the cytotoxic effect of doxorubicin on HCCLM3, SNU387, SNU49, and SK-Hep-1 hepatocellular carcinoma cells, which had a predominantly mesenchymal signature with low expression of E-cadherin but high expression of N-cadherin. Co-administration of ASC reduced doxorubicin-induced invasion/migration and modulated EMT characteristics in mesenchymal cells. This process was probably mediated by the E-cadherin repressors Snail and Slug. In addition, ASC increased sensitivity to doxorubicin treatment by directly inhibiting STAT3 binding to the Snail promoter. We also observed that ASC significantly enhanced the effect of doxorubicin against tumor growth and inhibited metastasis in an HCCLM3_Luc orthotopic mouse model. Collectively, our data demonstrate that ASC can increase sensitivity to doxorubicin therapy and reverse the EMT phenotype via the downregulation of STAT3-Snail expression, which could form the basis of a novel therapeutic approach against hepatocellular carcinoma. Mol Cancer Ther; 15(12); 2966-76. ©2016 AACR.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kβ silencing.

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kβ, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kβ kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kβ depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kβ inhibition. A small proportion of these tumors was resistant to PI3Kβ downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kβ dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kβ associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.

High expression of the Notch ligand Jagged-1 is associated with poor prognosis after surgery for colorectal cancer.

The importance of Notch signaling in colorectal cancer (CRC) carcinogenesis and progression has previously been presented. Increased expression of Jagged‐1 (JAG1), a Notch ligand, in CRC has been revealed, but the detailed prognostic significance of JAG1 in CRC has not been determined. Protein expression of JAG1 was examined using immunohistochemistry in 158 CRC specimens. Expression of JAG1 and E‐cadherin and their associations with clinicopathologic characteristics, overall survival (OS) and relapse‐free survival (RFS) were evaluated. In vitro studies using compounds to regulate intracellular signaling and small interfering RNA to silence JAG1 were performed in a colon cancer cell line. JAG1 expression in cancerous tissues was weak, moderate or strong in 32%, 36% and 32% of specimens, respectively, and correlated with histologic type and T stage. In multivariate analysis, JAG1 expression, histologic type and lymphatic invasion independently correlated with OS and RFS. The combination of high JAG1 expression and low E‐cadherin expression had an additive effect toward poorer OS and RFS compared with the low JAG1/high E‐cadherin expression subtype. A significant correlation between JAG1 expression and KRAS status was detected in groups stratified as high E‐cadherin expression. In vitro studies suggested that RAS‐MEK‐MAP kinase and the Wnt pathways positively regulated JAG1 expression. Gene silencing with siJAG1 indicated that JAG1 promotes the transition from epithelial to mesenchymal characteristics and cell growth. High expression of JAG1 is regulated by various pathways and is associated with poor prognosis through promoting the epithelial to mesenchymal transition and cell proliferation or maintaining cell survival in CRC.

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.

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions.

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of β1 integrin and low expression of E-cadherin. Addition of β1 integrin blocking antibody or siRNA-mediated downregulation of β1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and β1 integrin in EOC spheroid formation.

Expression of FoxM1 and the EMT-associated protein E-cadherin in gastric cancer and its clinical significance.

The aim of the present study was to investigate the expression of forkhead box M1 (FoxM1) and E-cadherin in tissues of gastric cancer in order to reveal any correlation between FoxM1, E-cadherin and clinicopathological parameters. The association between FoxM1 and E-cadherin in the development and progression of gastric cancer was also investigated. The expression of FoxM1 and E-cadherin in gastric cancer and adjacent normal tissue on tissue microarray was detected using immunohistochemistry. The clinicopathological significance of FoxM1 and E-cadherin in gastric cancer was explored, and the association between FoxM1 and E-cadherin was further examined using statistical techniques. In gastric cancer tissues, the expression of FoxM1 and E-cadherin was strongly positive, but it was weak in normal gastric mucosa. Overexpression of FoxM1 was evident in gastric cancer, and was associated with poor tumor differentiation (P<0.05), advanced tumor state (P<0.05) and lymph node (or distant) metastasis (P<0.05), whereas E-cadherin had the opposite effects. Furthermore, the correlation between FoxM1 and E-cadherin expression in gastric cancer tissue was negative. In conclusion, the high FoxM1 expression and low E-cadherin expression in gastric cancer tissue suggests that these proteins play a critical role in the development and progression of gastric cancer.

Neuropeptide Y expression in primary cutaneous melanoma.

Neuropeptide Y (NPY) is involved in the carcinogenesis of different tumours, especially neural crest-derived tumours. The aim of our study is to investigate the expression of NPY on melanoma and its relation with prognostic histological parameters and survival. This is a retrospective observational study of two independent series, with a total of 79 primary melanomas, diagnosed in two independent University Hospitals in Spain, from January 2000 to December 2004. We found a significant higher expression of NPY on superficial spreading melanoma and lentigo maligna (40%) (P = 0.030). Thinner tumours were associated with higher NPY expression (Clark level, P = 0.003; Breslow level, P = 0.012). Melanomas with low NPY expression were associated with intense cell proliferation (Ki-67, P = 0.034), high density of peritumoral mast cell infiltrates (P = 0.033) and low E-cadherin expression (P = 0.031). Melanomas with high NPY expression exhibited significant differences in terms of relapse time (median: 114 vs. 68 months, P = 0.008) and overall survival (114 vs. 74 months, P = 0.004). High expression of NPY was associated with better prognostic histological parameters, low peritumoral mast cells density, presence of adhesion proteins and better outcome.

Abstract 5050: The significance of the expression of E-cadherin and CD44 in patients with unresectable metastatic colorectal cancer

Abstract The loss of the adhesion molecules is reported to correlate with tumor invasion and metastasis in patients with various cancers. E-cadherin is an important molecule for cell-to-cell adhesion. While, CD44 is important for cell-to-extra cellular matrix adhesion. The aim of the present study is to evaluate the significance of the expression of E-cadherin and CD44 in patients with the unresectable metastatic colorectal cancer who are undergoing palliative chemotherapy. Formalin-fixed, paraffin-embedded samples were obtained from 49 patients who underwent primary tumor resection and who were receiving palliative chemotherapy for unresectable metastatic colorectal cancer. We evaluated the expression of E-cadherin and CD44 by immunohistochemistry. The expression of E-cadherin was not significantly associated with progression-free survival (PFS) or overall survival (OS). Although, the expression of CD44 did not correlate with PFS, it tended to correlate with the OS (p = 0.0699). According to the combination of CD44 and E-cadherin, both low expression group showed a significant poor PFS (p = 0.0101) and OS (p = 0.0009). Objective response rate and disease control rate were significantly decreased in both low expression group (p = 0.0076 and p = 0.0294, respectively). A univariate analysis to determine the variables that were associated with PFS indicated that the both low expression of E-cadherin and CD44 (p = 0.0474) and gender (p = 0.0330) were significantly associated with decreased survival. A multivariate analysis showed that the both low expression of E-cadherin and CD44 was an independent risk factor for decreased PFS (hazard ratio [HR]: 8.276, 95% confidence interval [CI]: 1.383-43.311; p = 0.0227). According to OS, the both low expression of E-cadherin and CD44 expression was revealed a prognostic factor by univariate and multivariate analysis(HR:15.118, 95%CI: 2.645-77.490; p = 0.0039). This study suggested that the combined low expression of both E-cadherin and CD44 was a strong independent predictor of decreased chemotherapeutic outcome and survival in patients with unresectable metastatic colorectal cancer. Citation Format: Yasuhito Iseki, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsuro Tamura, Go Ohira, Sadaaki Yamazoe, Katsunobu Sakurai, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. The significance of the expression of E-cadherin and CD44 in patients with unresectable metastatic colorectal cancer. [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 5050.

Abstract 2844: Cell senescence and antitumor potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids

Abstract Brazilian flora is considered one of the most diverse in the world and represents a source of new molecules with bioactivity against several diseases. 7-epiclusianone, a prenylated benzophenone was isolated from Garcinia brasiliensis, a plant named bacupari in folk medicine. Previous studies showed that this compound has dose-dependent cytotoxic effect in several cell lines derived from human cancers and antiproliferative effect by inducing cell cycle arrest in G1/S and apoptosis in A549 lung cancer cell line. The present study aimed to analyze the cytotoxic and/or antiproliferative potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids. Monolayer cell cultures are commonly used for testing drug effects largely because of their easy maintenance, but they do not represent the spatial interactions of cells within a tumor. Spheroids in 3D cell cultures can overcome some of those limitations thus mimicking the architecture of solid tumors. Initially the 3D conditions were established for both cell lines MCF7 and Hs578T. Spheroids were morphologically characterized by light and transmission electron microscopy. MCF-7 spheroids showed typical epithelial organization with cohesive cells, in accordance with higher expression levels of E-cadherin compared to monolayer. In Hs578T spheroids, cells assumed fibroblast-like morphology concentrically organized, low E-cadherin expression and synthesis of extracellular matrix components. The IC50 values of 7-epiclusianone were 20 μM for Hs 578T cells and 6 μM for MCF-7 monolayers cell cultures. At this concentration, the compound treatment arrested monolayer cell cultures in G0/G1. 7-epiclusianone reduced the mRNA levels for cyclins D1 and E in line MCF-7, while only cyclin E mRNA in Hs 578T. The compound did not change the microfilaments organization or the nuclear integrity, as observed by laser scanning confocal microscopy. Interestingly, 7-epiclusianone treated cultures exhibit higher senescence indexes while apoptotic cells were not detected. Altogether, these data suggest that 7-epiclusianone is a promising molecule against breast cancer cells. The three-dimensional culture was more resistant to treatment with the compound than the monolayer, therefore more comprehensive studies are needed to understand better the effects of 7-epiclusianone on this type of culture. (Supported by FAPESP and CNPq) Citation Format: Bianca Rocha-Sales, Paula Rezende-Teixeira, Evandro Luís Oliveira Niero, Camila Lauand, Marisa Ionta, Simone SL Hanemann, Glaucia M. Machado-Santelli. Cell senescence and antitumor potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids. [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 2844.

Biological characterization of bovine mammary epithelial cell lines immortalized by HPV16 E6/E7 and SV40T.

Primary bovine mammary epithelial cells are not ideal models for long-term studies, because primary cells undergo a limited number of proliferations in vitro and enter into a growth-arrest stage called cell replicative senescence; we therefore must establish the immortalized bovine mammary epithelial cells (BMECs) in vitro. More importantly, the mechanisms of the relationship between immortalized and apoptotic cell remain unknown in BMECs. We therefore sought to elucidate the mechanisms of which immortalized cells escape the pathway of apoptotic signal. These cells were successfully immortalized without any signs of senescence. The maximum number of BMEC and E6E7 immortalized cells were reached after 6d of culture. At this point, there were significantly more E6E7 immortalized cells than primary BMECs (P < 0.01). The population-doubling times of the E6E7 and SV40T immortalized cells were lowest at 48 and 72h. We failed to detect the expression of the epithelial cell marker E-cadherin in BMECs; however, immortalized cells had low expression of E-cadherin. The expression of β-catenin was markedly expressed in immortalized cells than in BMECs (P < 0.01). Caspase-3, caspase-9, and poly ADP-ribose polymerase (PARP) were detected; however, the cleavage of caspase-3 and PARP was not observed. Our data demonstrate that the expressions of caspase-9, caspase-3, and PARP are not sufficient for the apoptosis of immortalized cells and suggest that E-cadherin and β-catenin might be an important indicator of the development of cancer.

Parameters of Stromal Activation and Epithelial to Mesenchymal Transition as Predictive Biomarkers for Induction Chemotherapy in Patients With Locally Advanced Oral Cavity and Oropharyngeal Squamous Cell Cancer.

ObjectivesInduction chemotherapy (IC) is likely to be effective for biologically distinct subgroups of oral cancer and biomarker development may lead to identification of those patients.MethodsWe evaluated immune cell infiltration, stroma formation and structure of the invasive front as well as the immunohistochemical expression of alpha smooth muscle actin (ASMA), CD163, E-cadherin, N-cadherin, and the laminin gamma 2 chain in pretreatment biopsy specimens and surgical resections after IC in 20 patients with locally advanced oral cancer who were treated in a prospective, ongoing, phase II trial on IC using docetaxel, cisplatin, and 5-fluorouracil (TPF).ResultsSignificant negative prognostic factors for incomplete pathological tumor response to IC were alcohol abuse (P=0.032), cN+ (P=0.042), and <30% tumor reduction after first cycle of IC (P=0.034). Of the investigated histological parameters and biomarkers only a low membrane-bound expression of E-cadherin showed a trend to be associated with incomplete response to IC (P=0.061). Low expression of ASMA in stromal vessels and a strong tumor invasion front were significantly associated to tumor recurrence (P=0.024 and P=0.004, respectively). The median follow-up of all patients was 35 months. Alcohol abuse (P<0.001), <30% tumor reduction after first cycle of IC (P=0.005), and a strong tumor invasion front (P=0.019) were negative prognostic factors for overall survival.ConclusionA strong predictive biomarker among the investigated parameters for benefitting from TPF IC could not be found. The extent of the tumor invasion front was a negative prognostic marker for recurrence and survival in oral cancer treated by TPF IC followed by surgery and postoperative radiochemotherapy.

Association between cancer stem cell-like properties and epithelial-to-mesenchymal transition in primary and secondary cancer cells

One of the theories on cancer stem cells (CSCs) states that these cells initiate most tumors and give rise to more-or-less differentiated tumor cells. Genetic signatures of CSCs are thought to predict tumor recurrence and metastases, thus, supporting the notion that CSCs may be metastatic precursors and induce epithelial-to-mesenchymal transition (EMT). In this study, we tried to examine the association between CSCs and EMT (using specific markers) in the mucoepidermoid carcinoma cell line YD15 and its derivative cell line YD15M (lymph node metastasis). Relative protein expression levels were analyzed by western blotting, flow cytometry, and immunofluorescence assays. In addition, cell cycle assay and aldehyde dehydrogenase (ALDH) activity assay were carried out. Under growth conditions, YD15M cells formed irregular spherical colonies consistent with a stem cell phenotype. YD15M cells demonstrated the low expression of E-cadherin and β-catenin but high expression of vimentin than that in YD15 cells. In the metastatic cells (YD15M), the coexpression of vimentin and CD133 was detected. Weak proliferation based on cell cycle analysis and decreased PCNA expression was also observed. In addition, expression levels of ALDHA1, OCT4, and NANOG (CSC-like properties) were significantly increased in YD15M cells. Taken together, these findings should help to elucidate the interplay between EMT and CSC-like properties during metastasis and may provide useful information for the development of a novel classification system and therapeutic strategies against head and neck cancer.

Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer

BackgroundSmall cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and epithelial to mesenchymal transition have been associated with increased chemoresistance and metastatic spread in SCLC. Patients and MethodsThe biopsy specimens of 38 SCLC patients were used for marker evaluation by immunohistochemistry. The markers for CSCs were CD44 and SOX2. The markers for epithelial to mesenchymal transition were E-cadherin, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, vimentin, and c-MET. Staining was scored as low (weak) or high (strong) intensity for SOX2, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, and c-MET and using the immunoreactive score for CD44, E-cadherin, and vimentin, expressed as low or high expression. ResultsHigh expression of c-MET (c-METH) and low expression of E-cadherin (E-cadL) showed a trend toward a better prognosis (P = .07 and P = .09, respectively). The combination of c-METH and E-cadL resulted in significantly better survival (P = .007). The tested markers were not associated with CTCs, although a trend was seen for c-METHE-cadL (P = .09) with low CTCs. The CSC markers SOX2 and CD44 were not associated with overall survival in this patient cohort. ConclusionSCLC with a mesenchymal-like phenotype (c-METHE-cadL) is associated with longer survival and showed a trend toward lower CTCs.

Mimicking Metastases Including Tumor Stroma: A New Technique to Generate a Three-Dimensional Colorectal Cancer Model Based on a Biological Decellularized Intestinal Scaffold.

Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of β-catenin in a subset of cells. One central drawback of 2D cultures—especially in consideration of drug testing—is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context—especially for substances targeting tumor–stroma interactions.

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.

Prognostic and Functional Significance of MAP4K5 in Pancreatic Cancer.

ObjectivesMAP4K5 plays an important role in regulating a range of cellular responses and is involved in Wnt signaling in hematopoietic cells. However, its functions in human malignancies have not been studied. The major objectives of this study are to examine the expression, functions and clinical significance of MAP4K5 in pancreatic ductal adenocarcinoma (PDAC).Materials and MethodsThe expression levels of MAP4K5, E-cadherin, vimentin, and carboxylesterase 2 (CES2) were examined by immunohistochemistry in 105 PDAC and matched non-neoplastic pancreas samples from our institution. The RNA sequencing data of 112 PDAC patients were downloaded from the TCGA data portal. Immunoblotting and RNA sequencing analysis were used to examine the expression of MAP4K5 and E-cadherin in pancreatic cancer cell lines. The effect of knockdown MAP4K5 using siRNA on the expression of CDH1 and vimentin were examined by Real-time RT-PCR in Panc-1 and AsPC-1 cells. Statistical analyses were performed using IBM SPSS Statistics.ResultsMAP4K5 protein is expressed at high levels specifically in the pancreatic ductal cells of 100% non-neoplastic pancreas samples, but is decreased or lost in 77.1% (81/105) of PDAC samples. MAP4K5-low correlated with the loss of E-cadherin (P = 0.001) and reduced CES2 expression (P = 0.002) in our patient populations. The expression levels of MAP4K5 mRNA directly correlated with the expression levels of CDH1 mRNA (R = 0.2490, P = 0.008) in the second cohort of 112 PDAC patients from The Cancer Genome Atlas (TCGA) RNA-seq dataset. Similar correlations between the expression of MAP4K5 and E-cadherin were observed both at protein and mRNA levels in multiple pancreatic cancer cell lines. Knockdown MAP4K5 led to decreased CDH1 mRNA expression in Panc-1 and AsPC-1 cells. MAP4K5-low correlated significantly with reduced overall survival and was an independent prognosticator in patients with stage II PDAC.ConclusionsMAP4K5 expression is decreased or lost in majority of PDACs. The strong associations between low MAP4K5 expression and loss of E-cadherin, reduced CES2 expression and decreased overall survival may suggest an important role of MAP4K5 in epithelial-to-mesenchymal transition, chemotherapy resistance and tumor progression in pancreatic cancer. Targeting impaired MAP4K5 signaling may represent a new therapeutic strategy for pancreatic cancer.

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 P6-03-10: Genomic and transcriptomic heterogeneity in metaplastic breast carcinomas

Abstract Introduction: Metaplastic breast carcinoma (MBC) is a rare form of triple-negative breast cancer (TNBC), accounting for approximately 0.2%-5% of all invasive breast cancers. These tumors are characterized by the presence of neoplastic cells displaying differentiation towards squamous epithelium or mesenchymal elements. MBCs are reported to have an aggressive clinical behavior, to exhibit a worse prognosis and to respond less frequently to conventional chemotherapy regimens than common forms of TNBCs. In this study, we sought to define whether morphologically distinct subgroups of MBCs would be underpinned by distinct gene expression or copy number profiles, and whether MBCs, akin to other special histologic types of TNBC (e.g. secretory carcinoma and adenoid cystic carcinoma), would be underpinned by a highly recurrent fusion gene. Methods: RNA and DNA samples were extracted from microdissected frozen MBCs (5 squamous, 5 spindle and 7 chondroid) and subjected to gene expression profiling using the Illumina Human HT-12 v4 platform and gene copy number profiling using the Affymetrix Human SNP 6.0 arrays, respectively. Genes differentially expressed between MBC subtypes were identified using SAM, and functional annotation of these genes was performed using Ingenuity Pathway Analysis. Intrinsic molecular subtypes were determined using the PAM50 and claudin-low intrinsic gene lists. In addition, all cases were subjected to paired-end massively parallel RNA-sequencing (Illumina GAIIx). Putative expressed fusion transcripts were identified using a validated algorithm (i.e. ChimeraScan), and confirmed by means of RT-PCR. Results: MBCs with spindle cell morphology were all classified as of claudin-low intrinsic subtype, whereas MBCs with chondroid or squamous cell metaplasia were classified as of normal breast-like, basal-like or claudin-low subtypes, suggesting that these morphologic subgroups are heterogeneous. Unsupervised analysis of microarray and RNA-sequencing gene expression data further demonstrated that MBCs with spindle cell differentiation displayed distinctive transcriptomic profiles, and formed clusters distinct from those enriched for MBCs with chondroid and squamous cell metaplasia. MBCs with spindle cell morphology preferentially expressed regulators of epithelial-to-mesenchymal transition including lower expression of E-cadherin and EpCAM. At the genomic level, MBC subtypes displayed patterns of gene copy number alterations similar to those of common forms of TNBCs from The Cancer Genome Atlas, and no significant differences were found among the distinct MBC subtypes. Nine in-frame fusion genes, TBL1XR1-PIK3CA, WAPL-CDHR1, MAP2K3-HMGCLL, PARG-BMS1, FN1-ICAM1, TNKS1BP1-SPARC, AAK1-ARNT2, MBTPS1-TCEANC2 and PSMA6-SHMT1 were identified and validated in the index cases, however none of these was found to be recurrent in the cases analyzed in this study. Conclusion: MBC subtypes, despite harboring similar patterns of gene copy number alterations, display significant transcriptomic differences, which may account for their distinct histologic features. Our findings also demonstrate that unlike other histologic special types of TNBC, MBCs are not underpinned by a highly recurrent expressed fusion gene. Citation Format: Piscuoglio S, Ng CKY, Cowell CF, Mariani O, Martelotto L, Natrajan R, Lim RS, Maher CA, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Genomic and transcriptomic heterogeneity in metaplastic breast carcinomas. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-10.

DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing possibility that reactivation of E-cadherin expression through promoter demethylation may represent a potential therapeutic strategy for the treatment of melanoma.