Epithelial-mesenchymal Transition Phenomenon Research Articles

Abstract 911: Destabilizing β-catenin and Ras could be an ideal therapy for treating metastatic colorectal cancer

Abstract INTRODUCTION Aberrant activations of Wnt/β-catenin and Ras signaling pathways by APC (90%) and K-Ras (40-50%) mutations are closely interacted and accelerate CRC tumorigenesis. Protein level of β-catenin as well as Ras is increased by APC loss via inhibiting GSK3B-mediated poly-ubiquitination dependent Ras degradation through recruitment of β-TrCP E3 linker. Moreover, additional K-Ras mutation results in the liver metastasis by secondary activation of the Wnt/β-catenin signaling pathways via Ras-ERK and -AKT pathways in addition to the initial activation by APC loss. Consequently, both β-catenin and Ras were highly increased in human CRC patient tissues, especially in metastatic tumor. EXPERIMENT PROCEDURES To investigate the synergistic effects of aberrant Wnt/β-catenin and Ras signaling pathways on tumor development to metastasis, we established the APCMin/+/K-RasG12DLA2 compound mouse that harbor APC and K-Ras mutations in animal model. We also analyzed Ras protein level using human CRC patient tissues of normal mucosa and adenocarcinoma (N=24) and metastatic adenocarcinoma (N=26) and tumor budding (N=10). To recapitulate the metastatic tumor environment which have enriched receptor tyrosine kinases (RTKs), we maintained and experimented with EGF (20ng/ml) in every in vitro studies. RESULTS We observed that both β-catenin and Ras were increased in adenocarcinoma and metastatic adenocarcinoma of human CRC compared with normal mucosa. Interestingly, both β-catenin and Ras were most significantly increased in tumor budding regions which histopathologically represent epithelial mesenchymal transition (EMT). In murine model, the EMT phenomenon was effectively induced and invasion of small intestinal tumors were occurred in APCMin/+/K-RasG12DLA2 with increased level of β-catenin and Ras compared with APCMin/+. However, EMT phenomenon and its following events such as migration and invasion of CRC cells induced by APC and K-Ras mutations were abolished by destabilization of β-catenin and Ras. CONCLUSION Our results show that destabilizing β-catenin and Ras could be effective therapeutic targets for inhibiting metastasis in the early stage of CRC development Citation Format: Yong-Hee Cho, Jeong-Soo Yoon, Kang-Yell Choi. Destabilizing β-catenin and Ras could be an ideal therapy for treating metastatic colorectal cancer [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 911. doi:10.1158/1538-7445.AM2017-911

MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling.

TGF-β1-induced epithelial-mesenchymal transition (EMT) has been proved to be associated with metastasis of breast cancer cells. We attempted to detect a novel mechanism that microRNAs mediated the TGF-β1-induced EMT in the process of breast cancer metastasis. Here we reported that the expression of miR-23a was higher in breast cancer cells with high metastasis ability and patients with lymph node metastasis and the treatment of TGF-β1 significantly upregulated the expression of miR-23a in breast cancer cells. We found that miR-23a was upregulated by TGF-β1 post-transcriptionally and Smads directly bound the RNA Smad binding element (R-SBE) of miR-23a. Functional studies showed that inhibition of miR-23a suppressed the TGF-β1-induced EMT, migration, invasion and metastasis of breast cancer both in vitro and in vivo. In addition, we determined that miR-23a directly targeted and suppressed CDH1, one important gene in EMT phenomenon. Notably, Wnt/β-catenin signaling was activated by the suppression of CDH1 in the miR-23a mediated process of TGF-β1-induced EMT and tumor invasion. These results demonstrate that miR-23a promotes TGF-β1-induced tumor metastasis in breast cancer by targeting CDH1 and activating Wnt/β-catenin signaling. Taken together, our results indicate a novel regulatory mechanism of TGF-β1-induced EMT and suggest that miR-23a might be a potential target in breast cancer therapy.

CD24 expression is a marker for predicting clinical outcome and regulates the epithelial-mesenchymal transition in ovarian cancer via both the Akt and ERK pathways.

The degree of peritoneal dissemination and chemotherapy-resistant tumors is related to the prognosis in patients with advanced-stage ovarian cancer. The epithelial-mesenchymal-transition (EMT) is a multifaceted pathological program that endows cancer cells with the ability to invade and disseminate. CD24 is frequently overexpressed in various human cancers and is correlated with a poor prognosis. We herein examined the functions of CD24 in human ovarian cancer cell lines and evaluated how it contributes to the molecular mechanism underlying the regeneration of cancer stem-like cells (CSCs) through the EMT mechanism in ovarian carcinoma. We demonstrated that CD24 was expressed in 70.1% of primary ovarian carcinoma tissues, which were obtained from 174 patients, and that the expression of CD24 was an independent predictor of survival in patients with ovarian cancer. The expression of CD24 has been found to be correlated with the FIGO stage, presence of peritoneal and lymph node metastasis. CD24 induces the EMT phenomenon, which is involved in cell invasion, the highly proliferative phenotype, colony formation and which is associated with cisplatin resistance and the properties of CSCs, via the activation of PI3K/Akt, NF-κB and ERK in Caov-3 cisplatin-resistant cell lines. CD24-positive ovarian carcinomas have been shown to have a greater potential for intra-abdominal tumor cell dissemination in in vivo models. Our findings suggest that CD24 induced the EMT phenomenon in ovarian cancer, and that CD24 amplified cell growth-related intracellular signaling via the PI3K/Akt and MAPK pathways by affecting the EMT signal pathways. We believe that CD24 is a key molecule of metastatic progression in the EMT phenomenon and a promising therapeutic target for advanced ovarian cancer.

Abstract B64: A high-throughput screen identifies fulvestrant as an enhancer of immune-mediated cytotoxicity of mesenchymal-like lung cancer cells

Abstract The phenomenon of epithelial-mesenchymal transition (EMT) is a phenotypic switch that allows epithelial tumor cells to acquire features of mesenchymal cells, including the ability to migrate and invade and, therefore, to disseminate from the primary site. In addition to promoting tumor dissemination, various reports have demonstrated that tumor EMT also associates with the acquisition of resistance to the cytotoxic effects of chemotherapy, radiation, and some targeted therapies. In the present report we have utilized a quantitative high-throughput screening of a pharmaceutical collection of more than 2000 compounds to identify FDA-approved drugs capable of augmenting the sensitivity of mensenchymal-like lung cancer cells to immune-mediated lysis. Using this approach we have identified the estrogen receptor antagonist fulvestrant as capable of rendering mesenchymal-like lung cancer cells more susceptible to the cytotoxic effects of natural killer or antigen-specific T cells and chemotherapy. Fulvestrant-treatment of human lung carcinoma cell lines, both in vivo and in vitro, was associated with a reduction of mesenchymal features. In silico analysis of publicly available gene expression datasets revealed a novel association between the expression of mesenchymal-associated features and the expression of estrogen receptor alpha in lung adenocarcinoma patients. Although the potential role of estrogen signaling in lung cancer remains controversial, this study supports a positive association between active estrogen signaling and the promotion of a mesenchymal phenotype in lung cancer. The findings from this study form the rationale for future combinations of fulvestrant and immune-mediated therapies in the treatment of advanced lung cancer. Citation Format: Duane H. Hamilton, Lesley Mathews-Griner, Jonathan M. Keller, Xin Hu, Noel Southall, Juan Marugan, Justin M. David, Ferrer Ferrer, Jeffrey Schlom, Claudia Palena. A high-throughput screen identifies fulvestrant as an enhancer of immune-mediated cytotoxicity of mesenchymal-like lung cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B64.

Changes in mitochondrial function during EMT induced by TGFβ-1 in pancreatic cancer.

Mitochondrial dysfunction is linked to cancer. Differences in the number, morphology and function of mitochondria have been observed between normal cells and cancer cells. However, changes in mitochondrial function during epithelial-mesenchymal transition (EMT) in pancreatic cancer are less known. In the present study, the cultured human pancreatic cancer cell line Panc-1 was treated with transforming growth factor (TGF)β-1. Mitochondrial functions following TGFβ-1 exposure in pancreatic cancer were investigated. It was noticed that TGFβ-1 treatment induces morphologic changes and a shift from epithelial to mesenchymal phenotype in pancreatic cancer. Furthermore, increased mitochondrial mass was detected in pancreatic cancer following TGFβ-1 treatment. Besides, the production of reactive oxygen species in TGFβ-1-treated pancreatic cancer cells significantly increased compared with the control cells. Our results indicate that the phenomenon of EMT in pancreatic cancer has an association with mitochondrial dysfunction. Mitochondrial dysfunction may be a cause of EMT in pancreatic cancer, which leads to heterogeneity in pancreatic cancer, and may be a potential therapeutic target in the future.

Cisplatin promotes mesenchymal-like characteristics in osteosarcoma through Snail.

More than 30% of patients with osteosarcoma succumb to pulmonary metastases. Epithelial-mesenchymal transition (EMT) is a biological process by which tumor cells gain an increased capacity for invasiveness and metastasis. A previous study confirmed the phenomenon of EMT in osteosarcoma, a mesenchymal-derived tumor. However, whether chemotherapy affects EMT remains to be elucidated. In the present study, the osteosarcoma cells were exposed to a sublethal dose of cisplatin, and any surviving cells were assumed to be more resistant to cisplatin. In addition, these cells exhibited a more mesenchymal phenotype. Immunofluorescence analysis revealed that the cisplatin treated cells had an increased long/short axis ratio and increased expression of N-cadherin compared with control cells. A panel of EMT-associated genes was subsequently assessed by quantitative PCR and western blot analysis, and they were observed to be significantly upregulated in the cisplatin treated cells. The in vitro wound healing and Transwell assay indicated that the cisplatin treated cells were more prone to migrate and invade. An in vivo assay showed that the cisplatin-treated xenograft had increased expression of EMT-associated genes, and exhibited increased pulmonary lesions compared with the control, which indicated an elevated capacity to metastasize. The expression of Snail was knocked down by specific small interfering RNA, and it was observed that Snail inhibition promoted cisplatin sensitivity, and cisplatin-induced EMT was significantly blocked. Taken together, the results of the present study supported that idea that Snail participates in cisplatin-induced EMT in osteosarcoma cells, and targeting EMT-transcription factors may offer promise for the therapeutics of osteosarcoma.

Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics.

BackgroundSpheroid based culture methods are gaining prominence to elucidate the role of the microenvironment in liver carcinogenesis. Additionally, the phenomenon of epithelial-mesenchymal transition also plays an important role in determining the metastatic potential of liver cancer. Tumor spheroids are thus important models to understand the basic biology of liver cancer.MethodsWe cultured, characterized and examined the formation of compact 3-D micro-tumor spheroids in five hepatocellular carcinoma (HCC) cell lines, each with differing TP53 mutational status (wt vs mutant vs null). Spheroid viability and death was systematically measured over a course of a 10 day growth period using various assays. We also examined the TP53 and E-cadherin (CDH1) mRNA and protein expression status in each cell line of the 2-D and 3-D cell models.ResultsA novel finding of our study was the identification of variable 3-D spheroid morphology in individual cell lines, ranging from large and compact, to small and unstable spheroid morphologies. The observed morphological differences between the spheroids were robust and consistent over the duration of spheroid culture growth of 10 days in a repeatable manner. Highly variable CDH1 expression was identified depending on the TP53 mutational status of the individual HCC cell line, which may explain the variable spheroid morphology. We observed consistent patterns of TP53 and CDH1 expression in both 2-D and 3-D culture models.ConclusionsIn conclusion, we show that 3-D spheroids are a useful model to determine the morphological growth characteristics of cell lines which are not immediately apparent in routine 2-D culture methods. 3-D culture methods may provide a better alternative to study the process of epithelial-mesenchymal transition (EMT) which is important in the process of liver cancer metastasis.

Overexpression of long non-coding RNA-CTD903 inhibits colorectal cancer invasion and migration by repressing Wnt/β-catenin signaling and predicts favorable prognosis.

Accumulating evidence reveals that long non-coding RNA (lncRNA) is essential for tumorigenesis and progression, but little is known about its roles and mechanisms in metastatic colorectal cancer (CRC). This study aimed to detect expression level and prognostic role of lncRNA‑CTD903 in CRC patients, which was selected based on one microarray data. The effects on cell invasion, migration and proliferation were investigated after silencing or overexpression of CTD903 in CRC cell lines. We also observed the EMT (epithelial-mesenchymal transition) phenomenon and effect on cell adhesion. The associations between CTD903 and EMT markers, such as E-cadherin, N-cadherin, β-catenin, ZEB1, ZO-1, Snail, and Twist, were determined by western blotting. Our results showed lncRNA-CTD903 expression was strongly upregulated in 115CRC patients, comparing to adjacent normal tissues. CTD903 was proven to be an independent predicted factor of favorable prognosis in CRC patients by using multivariate Cox proportional hazards model. After knockdown of CTD903 in RKO and SW480, both cell invasion and migration increased, and cells exhibited EMT-like appearance, along with reduced adhering ability. Moreover, overexpression of CTD903 in DLD1 and HCT116 reversed these phenotypes. Furthermore, downregulation of CTD903 enhanced Wnt/β-catenin activation and subsequently increased transcription factors (Twist and Snail) expression, along with increased mesenchymal marker Vimentin and decreased epithelial marker ZO-1 level, while overexpressed CTD903 confirmed these associations. In conclusion, this study shows that LncRNA-CTD903 acts as a tumor suppressor in CRC and can inhibit cell invasion and migration through repressing Wnt/β-catenin signaling, which plays important roles in EMT and CRC metastasis.

Increased chemoresistance via Snail-Raf kinase inhibitor protein signaling in colorectal cancer in response to a nicotine derivative.

A tobacco-specific component, 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK), is a major risk factor for many cancers. Recent reports have demonstrated that NNK exposure may be associated with tumor progression and chemoresistance in certain cancers. However, the underlying NNK-induced mechanism contributing to the aggressiveness of colorectal cancer (CRC) has not been thoroughly studied. In this study, we used HT29 cells treated with NNK to simulate the long-term exposure of cigarette smoke. A comparative analysis was performed to evaluate cell proliferation, migration, and invasion as well as epithelial-mesenchymal transition (EMT) markers and drug-resistance genes expression, cancer stem cell (CSC) properties, and anti-apoptotic activity. Signaling pathways related to chemoresistance were also investigated. As a result, NNK exposure dose-dependently stimulates cell proliferation, enhance abilities of migration and invasion, induce EMT phenomenon, and attenuate apoptosis. Furthermore, NNK exposure also promotes the capabilities of sphere formation, upregulation of Snail, and overexpression of CD133, Nanog, OCT4, and the drug-resistant genes. Knockdown of Snail results in upregulation of Raf kinase inhibitor protein (RKIP), increased apoptosis, reversal of EMT phenomenon, and reducation of expression of CSC markers, all of which contribute to a decrease of chemoresistance. Our study demonstrates a number of related mechanisms that mediate the effect of NNK exposure on increasing CRC therapeutic resistance via the Snail signaling pathway. Targeting Snail may provide a feasible strategy for the treatment of CRC.

High Infiltration of Tumor-Associated Macrophages Influences Poor Prognosis in Human Gastric Cancer Patients, Associates With the Phenomenon of EMT.

Tumor-associated macrophages (TAMs) are associated with poor prognosis in numerous human cancers and play important roles in tumor progression. Epithelial-mesenchymal transition (EMT) contributes to invasion and metastasis in cancer. However, the associations between TAMs and EMT are not clear in gastric cancer (GC). The present study was designed to investigate the effects of TAMs on EMT in human GC.TAMs marker CD68 and EMT-related proteins were detected by immunohistochemistry (IHC) in human GC tissues and their clinical significance were evaluated.A high level of infiltration of TAMs was associated with aggressive characteristics of tumor and an independent poor prognostic factor in human GC tissues. Infiltration of TAMs was also associated with EMT-related proteins in human GC tissues.Our findings suggest that the high level of infiltration TAMs was associated with aggressive features of GC and is an independent poor prognostic factor in GC patients. TAMs are associated with EMT induction in human GC tissues. The level of TAMs infiltration may be used as a prognostic factor and even a therapeutic target in GC.

Cellular and molecular aspects at invasive tumor front in oral squamous cell carcinoma (part-I)

Oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity. Despite considerable advances in diagnostic and therapeutic field, its prognosis remains poor. Grading by histopathological features does not influence prognosis until deeply invasive margins are evaluated by pathologists. The most invasive part of malignant tumor reflects disturbance in molecular mechanism, alteration in gene expression that controls cell differentiation, growth, death (apoptosis) as well as interactions between cancer cells and stromal components that may lead to the formation of oncofetal extracellular matrix, enabling tumor cell phenotype transition and migration. In recent years, analysis of cell proliferation, DNA content, and epithelial-mesenchymal transition (EMT) phenomenon has been used in an attempt to identify new prognostic indicators. Great interest is also shown in matrix metalloproteinase which plays a key role in invasion and metastasis. This review focuses on altered pathways in OSCC at gene expression and protein level, role of proteinases and EMT phenomenon in tumor progression, invasion, and metastasis with respect to invasive tumor front.

Epithelial-mesenchymal axis in head and neck cancer cell lines

Objectives: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumour type which necessitates multiple invitro models to attain an appreciation of its multiple subtypes. The phenomenon of epithelial-mesenchymal transition (EMT) isimportant to the development of a metastatic cancer cell phenotype being relevant to the ability of cancer cells to intravasate intovasculature and to invade tissues. The role of EMT in human papilloma virus (HPV) positive HNSCC is not well understood. Thispaper aims to characterize seven HNSCC cell lines (FaDu, SCC-25, SCC-15, CAL27, RPMI2650) including two new HPV-16positive HNSCC cell lines (UD-SCC2, 93-VU-147T) for their epithelial and mesenchymal properties. Materials and methods: A panel of HNSCC cell lines from multiple head and neck anatomical sites were profiled for basalexpression of epithelial and mesenchymal characteristics at mRNA, protein and functional levels (proliferative, migratory andinvasive properties). Furthermore, 3D spheroid forming capabilities were investigated. Results: We found that the HPV-16 positive cell line, in particular UD-SCC2 demonstrated a more invasive and mesenchymalphenotype at the molecular and functional levels suggesting HPV infection may mediate some of these cellular properties.Moreover, HPV-negative cell lines were not strictly epithelial presenting with a dynamic range of expression. Conclusions: This study presents the molecular and phenotypic diversity of HNSCC cell lines. It highlights the need formore studies in this field and a scoring system where HNSCC cell lines are ranked according to their respective epithelial andmesenchymal nature. This data will be useful to anyone modelling HNSCC behaviour, providing a molecular context which willenable them to decipher cell phenotypes and to develop therapies which block EMT progression.

Abstract CN04-01: Targeting the epithelial-mesenchymal transition via brachyury-based cancer vaccines

Abstract Carcinoma cells undergo profound phenotypic changes during progression towards metastasis. One such phenotypic modulation is the epithelial-mesenchymal transition (EMT), an embryonically relevant process that can be reactivated in tumor cells, resulting in the acquisition of metastatic propensity, stem-like cell properties and resistance to a variety of anti-cancer therapies, including chemotherapy, radiation and some small molecule targeted therapies. Targeting of EMT is now emerging as a novel intervention against tumor progression. Brachyury, a transcription factor of the T-box family, is a driver of the phenomenon of EMT in human carcinomas. High levels of brachyury have been demonstrated in several human tumors, including chordoma, hemangioblastoma, and a range of human carcinomas such as lung, breast, colon and prostate, while brachyury is absent in the majority of adult normal tissues. In preclinical studies, brachyury has been shown to induce expression of molecules associated with the mesenchymal phenotype in carcinoma cells, to promote cell motility and invasiveness in vitro, and to favor metastatic dissemination in xenograft models. In addition to its role in tumor EMT, several studies have now also shown that the expression of brachyury is a predictor of poor prognosis in lung, breast, hepatocellular and prostate cancer, and it has been recently suggested to play a predominant role in triple negative vs. non-triple negative breast tumors. We propose the use of cancer vaccines that target brachyury as a strategy to directly eradicate tumor cells undergoing the phenomenon of EMT. Brachyury fulfills two major requirements to be used as a target for vaccine approaches: (a) is a highly tumor restricted molecule, and (b) is highly immunogenic, as demonstrated by the ability to expand brachyury-specific cytotoxic T lymphocytes from the blood of cancer patients which, in turn, can lyse tumor cells that express the brachyury protein. Based on these observations, our laboratory has collaboratively developed two brachyury-based cancer vaccines that are currently undergoing clinical evaluation. A recombinant yeast-brachyury vaccine expressing the full-length brachyury protein has demonstrated anti-tumor activity in murine models in the absence of toxicity. A Phase I clinical trial of this vaccine has now been completed in patients with advanced carcinomas or chordoma, demonstrating ability to induce multifunctional, brachyury-specific CD8+ and CD4+ T-cell responses in patients post- vs. pre-vaccination, with some evidence of disease control in absence of toxicity. In addition, a recombinant poxviral vaccine encoding brachyury and three T-cell costimulatory molecules, designated MVA-brachyury-TRICOM, is currently undergoing Phase I clinical testing in patients with advanced tumors. To our knowledge, yeast-brachyury and MVA-brachyury-TRICOM are the first vaccines targeting a driver of the phenomenon of EMT that have successfully entered clinical development. Phase II studies with these vaccines are now ongoing and planned. Multiple preclinical studies are being conducted to optimize the combinatorial use of these vaccines with other anti-cancer agents, including checkpoint inhibitors and other agents. We hypothesize that eradication of brachyury-expressing cancer cells will result in clinical benefit by reducing the number of tumor cells with stem-like characteristics, including invasive/metastatic potential and resistance to conventional therapies.

Abstract A42: MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition

Abstract The phenomenon of epithelial-mesenchymal transition (EMT) is a phenotypic switch that allows epithelial tumor cells to acquire features of mesenchymal cells, including the ability to migrate and invade and, therefore, to disseminate from the primary site. In addition to promoting tumor dissemination, various reports have demonstrated that acquisition of features of EMT associates with tumor resistance to the cytotoxic effects of chemotherapy, radiation, and some targeted therapies. It is not well understood, however, the contribution of EMT to the escape of tumors from host immune-surveillance and immune-mediated rejection. Our laboratory has characterized the T-box transcription factor brachyury as a tumor-associated antigen and a regulator of EMT in human carcinomas. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrated that high levels of brachyury significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells, and render tumor cells less responsive to a cancer vaccine in syngeneic murine tumor models. This resistance was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon to loss of cell cycle kinase CDK1, which mediates lamin phosphorylation. In support of a causal connection, pre-treatment of tumor cells with MK-1775, a specific inhibitor of WEE1, which is known to be a negative regulator kinase of CDK1 activity, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggest reconstituting CDK1 activity to threshold levels might be sufficient to elevate immune-mediated lysis of mesenchymal-like cancer cells, and improve the efficacy of anti-tumor vaccines. Citation Format: Duane H. Hamilton, Romaine I. Fernando, Kwong-Yok Tsang, Claudia Palena. MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A42.

Abstract 756: Inhibition of ABCB1 overcomes cancer stem cell-like properties and acquired resistance to MET inhibitor in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) patients with EGFR mutations have shown a dramatic response to EGFR inhibitors (EGFR-TKI). EGFR T790M mutation and MET amplification have been recognized as mechanisms of acquired resistance to EGFR-TKI. Recently, MET inhibitors have been used in NSCLC patients for clinical trials. In this study, we tried to identify the mechanisms of acquired resistant to MET inhibitor. We analyzed the antitumor effects of two MET inhibitors; PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC1 cells with MET amplification were only sensitive cells to both of MET inhibitors. We have established PHA-665752 resistant cells; namely EBC1-R cells. EBC1-R cells showed high expression levels of ATP-binding Cassette Sub-family B Member 1 (ABCB1) as wells as phosphorylation MET (p-MET). EBC1-R cells grew as cell sphere that exhibited cancer stem cell-like (CSC) properties and epithelial mesenchymal transition (EMT) feature. Two miRNAs, miR-374a and miR-138 which targeted ABCB1, were decreased in EBC1-R cells. ABCB1 inhibitor elacridar could suppress sphere numbers and EMT phenomenon and restore to the resistance to PHA-665752 in EBC1-R cells. Our study demonstrated that ABCB1 overexpression associated with CSC properties and EMT may be critical for acquire resistance for MET inhibitor. ABCB1 inhibition might be a novel therapeutic strategy for resistance to MET inhibitor in NSCLC cells. Citation Format: Teppei Sugano, Masahiro Seike, Rintaro Noro, Chie Soeno, Shinji Nakamichi, Nobuhiko Nishijima, Masaru Matsumoto, Susumu Takeuchi, Akihiko Miyanaga, Kaoru Kubota, Akihiko Gemma. Inhibition of ABCB1 overcomes cancer stem cell-like properties and acquired resistance to MET inhibitor in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2015-756

Abstract 5016: Short-term blockade of EGF signaling improves immune-mediated cytotoxicity of NSCLC cells

Abstract One of the manifestations of tumor plasticity is the acquisition of mesenchymal-like features by epithelial tumor cells via the phenomenon of epithelial-mesenchymal transition (EMT). This phenotypic conversion of tumor cells is known to be associated with tumor resistance to multiple cancer therapies, including chemotherapy, radiation and some small molecule-targeted therapies. Recent work from our group demonstrated that tumor cells undergoing the EMT may also become resistant to immune-mediated cytotoxicity involving antigen-specific and innate effector cells. As signaling through the epidermal growth factor receptor (EGFR) is a major inducer of EMT in epithelial cells, in the present study we sought to investigate the effect of short-term EGFR inhibition with erlotinib on tumor phenotype, and provide rationale for combination with immune-mediated therapies. Our data shows that pretreatment with the EGFR TKI erlotinib for less than 72 hours modulates tumor plasticity and immune-mediated cytotoxicity in NSCLCs harboring a sensitizing EGFR mutation. A significant modulation in expression of multiple EMT markers can be observed, including significant changes in the levels of expression of the transcription factor brachyury, a driver of human tumor EMT and a target for immunotherapeutic interventions. In addition, significant modulation of immune-relevant molecules on the surface of tumor cells can be observed, including changes in the levels of TRAIL-receptors, co-stimulatory and checkpoint molecules. The observed phenotypic changes in response to short-term erlotinib treatment correlate with an enhancement of immune-mediated tumor cell lysis, without any observed adverse affect on effector cell populations. Taking into account that brachyury-based cancer vaccines are currently being evaluated in clinical trials, modulation of brachyury levels and the observed enhancement of immune-mediated lysis in erlotinib-treated tumors may provide the rationale for future combinations of erlotinib and brachyury-based vaccines for the treatment of lung carcinomas. Citation Format: Charli Dominguez. Short-term blockade of EGF signaling improves immune-mediated cytotoxicity of NSCLC cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2015-5016

Expression of adhesion molecules and epithelial-mesenchymal transition factors in medullary carcinoma of the colorectum.

Medullary carcinoma (MC) of the colorectum is known as a rare variant with favorable prognosis despite its poorly differentiated morphology. The mechanism of its favorable behavior has been unclear. Here, we compared the expressions of adhesion molecules and epithelial-mesenchymal transition (EMT)-related proteins in the central portion and invasive front between 43 MCs and 30 poorly differentiated adenocarcinomas (PDAs). The expressions of membranous E-cadherin (P < .0001), β-catenin (P < .0001) and claudin 1 (P = .0036) were significantly preserved in the invasive front of the MCs compared to those in the invasive front of the PDAs. E-cadherin membranous expression was also significantly preserved in the central portion of the MCs (P = .0178). Nuclear β-catenin expression in both the central portion (P = .0463) and invasive front (P = .0346) of the MCs was significantly less frequent compared to that in the PDAs. Snail (P = .0035) and Twist1 (P = .0463) expressions in the invasive front of the MCs were significantly less frequent compared to the PDAs, suggesting that the EMT phenomenon may occur rarely in colorectal MC. Reduced membranous E-cadherin expression in the MC central portion was significantly correlated with poor clinical outcome (P = .0086). Our immunohistochemical results indicate that preserved adhesion molecule protein and less frequent expression of EMT-related transcription factors in the invasive front contribute to the favorable prognosis of colorectal MCs. We suggest that a reduced expression of E-cadherin in the central portion might be a good biomarker for an unfavorable prognosis in cases of MC.

Abstract P2-07-06: Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer

Abstract Background: The Src pathway is known to regulate tumor matastasis and plays a role in epithelial-mesenchymal transition(EMT). Lapatinib, although, has improved clinical outcome for HER2 positive breast cancer patients, acquired resistance to lapatinib remains an important reason influencing its clinical efficacy. Our previous research shows lapatinib acquired resistance HER2 positive breast cancer cell lines, SKBR3-R and BT474-R have EMT phenomenon and multiple pathway activated. Niclosamide is an FDA-approved antihelminthic agent, which is found has cytotoxicity effect on tumor stem cells recently. We wished to determine the effect of niclosamide on EMT and lapatinib resistance cells and investigate niclosamide as a potential therapeutic agent for HER2 positive breast cancer. Methods: SKBR3 and BT474, two HER2 positive breast cancer cell lines, were continuously exposed to increasing doses of lapatinib to establish two stable cell lines resistant to lapatinib, SKBR3-R and BT474-R. Cell proliferation was determined by CCK8 assay. Protein expression was determined by western-blotting. Invasion ability was analyzed by transwell assay. FITC staining flow cytometry (FCM) was conducted to observe the percentage of apoptosis. Results: Both two HER2 positive lapatinib resistant cell lines, SKBR3-R and BT474-R had EMT phenomenon. Niclosamide had a stronger inhibition effect on lapatinib resistant cell lines than parental cell lines, and induced more apoptosis by FCM. Western-blot showed niclosamide could reverse the EMT phenomenon of SKBR3-R and BT474-R with E-cadherin up-regulated and snail,vimentin down-regulated at the concentration of 0.5-1μM. A significant reduction of Src signaling was also confirmed, as well as the downstream Akt and Erk pathway. After adding niclosimide for 48 hours, SKBR3-R and BT474-R’s capability of invasion were inhibited. Conclusion: Our results suggested that niclosamide had a strong cytotoxic effect on HER2 positive lapatinib resistant breast cancer cell lines. The EMT induced by lapatinib resistance could be reversed by niclosamide, associated with the inhibition of Src pathway activation, as well as the downstream pathways. Niclosamide treatment produced reduced levels of invasion, serving as a novel therapeutic way for lapatinib-resistant breast cancer patients. Citation Format: Junjun Liu, Xiaosong Chen, Yan Mao, Kunwei Shen. Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-06.

Abstract P4-04-11: Comparing tissue compositions of within-individual mammographically high and low dense breast tissue

Abstract Abstract Purpose Mammographic density (MD) refers to the extent of radio-opaque breast tissue on a mammogram. Adjusted for a women’s age and body mass index (BMI), it is a strong and independent risk factor for breast cancer (BC). The presentation of mammographically dense breast is not uncommon in the normal female population, and patients’ awareness of its association with BC risk has been growing following mandatory reporting of MD by physicians in several states of America. Women with breasts that have over 75% dense tissue are 4 to 6 times more likely to develop BC than women with breasts that have less than 10% dense tissue. However, the biological basis of how high MD raises BC risk remains elusive. We aimed to examine the histological and molecular differences between high and low dense breast tissues of healthy women, using specimens accrued from prophylactic mastectomy procedures. Method 48 women between 2008 and 2013 underwent prophylactic mastectomy at St Vincent’s Hospital and Peter MacCallum Cancer Centre due to a high BC risk profile. Of these, 41 were eligible for analyses. Tissue slice resected from the mastectomy specimen was X-rayed, and high (HD) and low dense (LD) regions were dissected based on the radiological appearance. The histological composition, immunohistochemistry and proliferation status were assessed on matched high and low MD tissue of the same breast. Signed rank test and paired t test were used for quantitative analyses of potential differences between HD and LD tissue. Result HD tissue demonstrated a significantly greater proportion of stroma (p&amp;lt;0.0001) and epithelium (p&amp;lt;0.0001), and less amount of fat (p&amp;lt;0.0001) than LD tissue (n=41 women). Epithelium from HD region also demonstrated epithelial-mesenchymal transition (EMT) plasticity, which was evident as the co-expression of cytokeratin (CK)-19 and vimentin in the glandular area. There was no significant difference with regards to oestrogen receptor (ER) (p= 0.2772), progesterone receptor (PR) (p= 0.9910), and Ki-67 (p= 0.6028) expression between HD and LD tissue. Conclusion and Significance We found that increased stroma and epithelium proportions contribute to the dense appearance on mammogram. Moreover, dense tissue did not demonstrate differed hormonal receptor expression or proliferation status from non-dense tissue, but showed a preponderance of EMT in the form of co-localisation by both CK-19 and vimentin in some of the epithelial cells. Our study is the first to report EMT phenomenon in benign mammary tissue, and suggests that investigations of the stromal micro-environment, and their interactions with epithelium may be key to improving our understanding on MD-mediated BC risk. Citation Format: Cecilia W Huo, Dexing Huang, Grace L Chew, Prue Hill, Wendy V Ingman, Michael Henderson, Kara L Britt, Rik Thompson. Comparing tissue compositions of within-individual mammographically high and low dense breast tissue [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-11.

Current research of epithelial-mesenchymal transition

Epithelial-mesenchymal transition（ EMT） refers to the transdifferentiation from epithelial cells to mesenchymal cells. The initial understanding of EMT is originated from the research on embryonic development,followed by the discovery of EMT phenomenon in physiological and pathological process in multicellular organism. In recent years,the occurrence and development of infiltration and metastasis of tumor cells along with fibrotic diseases that related to EMT are becoming the hot researches nowadays. Key words: Epithelial-mesenchymal transition; Embryonic development; Fibrotic disease; Tumor