Platelets at the Crossroads of Cancer: Activating Epithelial Mesenchymal Transition in Colorectal Carcinogenesis via Snail1

MP04-16 EPITHELIAL TO MESENCHYMAL TRANSITION PROMOTES PD-L1 EXPRESSION IN RENAL CELL CARCINOMA

Backgrounds: The epithelial to mesenchymal transition (EMT) is well known to play a crucial role in cancer invasion and metastasis and has been actively investigated in colorectal cancer (CRC). In recent years, the isoform switch of CD44, which is recognized to be a cancer stem cell marker, has been reported to associate with EMT, especially in breast cancer and hepatocellular carcinoma. However, the clinical impact of CD44 isoform switch to EMT in CRC is still unknown. Methods: E-cadherin, vimentin, CD44 standard (CD44s) and CD44 variant9 (CD44v9) expression were measured in 14 CRC cell lines and 150 CRC patients by real-time PCR. EMT status and CD44 status was determined by calculating vimentin/E-cadherin and CD44s/CD44v9 expression ratio, respectively. The CRC cell lines and patients were divided into two groups based on the EMT and CD44 status. We examined the association between the EMT status and CD44 isoform switch, and analyzed the correlation with clinicopathological factors and prognosis. Finally, we evaluated the effect of CD44 knockdown by siRNA in CRC cell lines by measuring the proliferation, migration and invasion ability. Results: 1. The CRC cell lines were classified into 8 epithelial, 4 mesenchymal and 2 intermediate type. Of these cell lines, CD44s was highly expressed in mesenchymal type cell lines, whereas, CD44v9 was highly expressed in epithelial type cell lines. 2. The 150 CRC patients were divided into 115 epithelial group and 35 mesenchymal group based on vimentin/E-cadherin expression ratio. Mesenchymal group showed significantly poorer survival than epithelial group (5-year survival rate; epithelial: 85.5% vs. mesenchymal: 62.1%, P = 0.009). EMT status was significantly correlated with invasion depth of tumor (P = 0.036), lymphatic vessel involvement (P = 0.034) and histological grade (P = 0.001). 3. High CD44 status group showed poorer prognosis than low CD44 status group (5- year survival rate; low group: 85.0% vs. high group: 62.1%, P = 0.025). There was a significant correlation between EMT status and CD44 status (r = 0.298, P < 0.001). On multivariate analysis, pathological T4 stage (P = 0.011), liver metastasis positive (P = 0.001), high CA19-9 level (≥37) (P = 0.003) and CD44 status (P = 0.001) were independent prognostic factors for CRC patients. 4. In COL-3-JCK, which is identified to be mesenchymal type, CD44 knockdown by siRNA decreased the vimentin expression at protein level. When the functional analysis were performed, CD44 knockdown significantly reduced the abilities of cell proliferation (P < 0.001), migration (P = 0.004) and invasion (P = 0.025). Discussion: EMT was a critical prognostic factor and CD44 isoform switch might contribute to EMT induction in CRC. These results could support a better understanding of the relationship between EMT and cancer stem cell. Therefore, further investigation of the specific splice isoform of CD44 might lead to novel therapeutic intervention for CRC patients. Citation Format: Naoki Mashita, Suguru Yamada, Naoki Iwata, Mitsuro Kanda, Daisuke Kobayashi, Chie Tanaka, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yashuhiro Kodera. Epithelial to mesenchymal transition might be induced via CD44 isoform switch in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1148. doi:10.1158/1538-7445.AM2014-1148

The ceRNA network regulates epithelial-mesenchymal transition in colorectal cancer

The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable 'CMS4 colorectal cancer molecular subtype' is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients' outcomes.

Purpose: African-American (AA) breast cancer (BC) and colorectal cancer (CRC) patients experience higher mortality rates than Non-Hispanic Caucasian (CA) patients. These disparities are thought to be due to race/ethnicity-specific tumor molecular biology. Our initial gene expression investigations have identified a Beta B2-Crystallin (CRYBB2) gene that is preferentially over-expressed in CRC of AA patients. However, the precise role of CRYBB2 in aggressiveness of CRC and BC has not been examined. Therefore, we hypothesize that biological mechanism linked to CRYBB2 could contribute to the disparate progression of CRC and BC in AA patients. Experimental Design: Gene expression studies were performed using Affymetrix Human Genome U133 Plus 2.0 arrays and mRNA samples extracted from 10 fresh frozen Stage III CRCs (5 from AA and 5 from CA) that were previously analyzed for the microsatellite instability (MS) status. Established CRC (HT29 and SW480) and BC (MBA-MD 231) cell lines were used to examine the phenotypic expression of CRYBB2. Also, paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft were assessed for CRYBB2 by western blot and immunofluorescence (IF). To study the involvement of CRYBB2 in CRC and BC biology, siRNA mediated gene silencing was used to inhibit CRYBB2 expression. The apoptosis of CRC and BC cells after knockdown of CRYBB2 was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The epithelial-mesenchymal transition (EMT) of CRC and BC cells was analyzed by western blot for E-cadherin, MM9 and vimentin expression. Western blot analysis was performed on lysates of CRC and BC cells to assess the effect of CRYBB2 knockdown for expression/ activation of the AKT, and mitogen-activated protein kinase pathway proteins. Results: Our microarray analyses have suggested that several key genes involved in tumor metastasis (CRYBB2, VSNL-1and PFN2) are up-regulated in MS-CRCs of AA but down regulated in MS-CRCs of CA patients. The phenotypic expression of CRYBB2 was found to be higher in CRC tissues as compared to matching normal tissues. Over-expression of CRYBB2 correlated with metastatic CRC and N-cadherin expression and the expression of CRYBB2 was substantially increased in metastatic lesion of CRC and BC xenografts as compared to the primary tumors. Knockdown of CRYBB2 induced apoptosis in CRC and BC cells. In addition, Knockdown of CRYBB2 suppressed EMT signatures, such as decreasing the expression of MMP9, vimentin (mesenchymal markers) and increasing the expression of E-cadherin (epithelial marker). Furthermore, depletion of CRYBB2 decreased the phosphorylation of AKT (p-AKT) in CRC and BC cells. CRYBB2 was mainly localized in cytoplasm of BC and CRC cells. Conclusions: Our data suggest that CRYBB2 inhibits apoptosis, activates EMT process and AKT phosphorylation by which it contributes the aggressiveness of CRC and BC. It may serve as a promising biomarker for identifying CRC and BC patients at high risk for metastases. It may also be useful as a therapeutic target to inhibit the growth and metastasis of CRC and BC. These studies are supported by NIH/NCI funds (R21-CA171251 and U54- CA 118948). Citation Format: Harvey L. Bumpers, Venkat Katkoori, Dongquan Chen, Upender Manne. The role of BetaB2-crystallin in progression of colorectal and breast cancers in African Americans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C56. doi:10.1158/1538-7755.DISP13-C56

Lung cancer accounts for the largest number of cancer-related deaths worldwide. Despite a new more discriminatory staging system for predicting survival, great variation in outcome within stage persists. Gene expression arrays and microRNA signatures from surgically resected primary tumors can predict survival and drug sensitivity within stage I disease better than standard staging protocols. Genes associated with epithelial mesenchymal transition (EMT) contribute to the malignant phase of tumor cell growth by controlling the switch to a metastatic phenotype. We hypothesize that the analysis of expression of EMT markers in thoracic lymph nodes may predict survival outcomes and drug responses in lung cancer patients. Endobronchial ultrasound fine needle aspiration (EBUS-FNA) of lymph nodes was performed on patients with known or suspected lung cancer to diagnose and/or stage disease. Total RNA was isolated from the FNA specimens, reverse transcribed, and analyzed for expression of a panel of genes associated with EMT using a probe-based real-time quantitative polymerase chain reaction (pqRT-PCR). Samples were analyzed for the expression of the epithelial markers cytokeratin 17 (CK17) and cytokeratin 19 (CK19) and the EMT markers Ecadherin (ECAD), Ncadherin (NCAD), and transcription factors snail, slug, ZEB1 and ZEB2. Samples from thirty-seven patients were processed for the study. Patients with metastatic disease had higher levels of snail and NCAD and lower levels of slug and ZEB2 compared to node negative and node positive patients, while patients with node negative disease had higher slug and lower levels of NCAD compared to node positive patients. ECAD did not vary by node positivity or presence of metastasis. The patients with adenocarcinoma on cytology showed higher levels of ECAD and snail, while patients diagnosed with squamous cell carcinoma had higher levels of ZEB1. In contrast, two patients with benign disease had low levels of all markers. Four patients with cytopathologically negative nodes expressed the epithelial markers CK17 and CK19, which are not typically present in lymph nodes. In limited follow-up, two patients with cytopathologically negative lymph nodes but high levels of ZEB1 had progression of disease. From this pilot study we have shown that EBUS-FNA consistently provides adequate samples from which RNA can be isolated and that genes associated with EMT can be used to identify cancer cells that have migrated to adjacent lymph nodes. We plan to enroll a total of 100 patients and follow them for 2 years to determine if this technique can identify patients at higher risk of metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3305.

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related death in the United States, and its development is closely correlated with lifestyle factors, including a high cholesterol “Western” diet. In our novel study, we investigated the etiology of dysregulated cholesterol homeostasis in CRC and characterized its systemic effects on tumor progression. Methods: Expression of DHCR7, the rate-limiting enzyme of cholesterol biosynthesis, in CRC was analyzed in silico using TCGA-COAD data. A human tissue microarray (n=208 cores) with samples from normal colon, local colon tumors, and lymph node metastases was stained for DHCR7 using IHC. Stained tissue cores were scored blindly by a clinical pathologist based on our histological scoring system, which accounted for the percentage of positive cells and the intensity of staining. We generated an APCCDX-CreER CRC mouse model and collected colonic tissue samples for analysis. Tissues were stained for DHCR7 and scored. To assess the impacts of high diet cholesterol on epithelial-to-mesenchymal transition (EMT) in vivo, we developed an APC model which was divided into three diet groups of equal size: a control group (fed a 0 ppm cholesterol diet), a low cholesterol group (186 ppm), and a high cholesterol group (880 ppm). Colonic tissue samples were stained for claudin-1, a classical marker of epithelial barrier integrity. MTT assays were used to measure the in vitro proliferation of SW620 human CRC cells incubated in 0, 5, 10, 15, 20, and 25 μM cholesterol. Results: In silico data analysis indicated that DHCR7 is broadly overexpressed in CRC patients. Histological scoring of human patient samples showed upregulation of DHCR7 in local and metastatic CRC tumors compared to normal colonic tissue. Surprisingly, DHCR7 expression was significantly higher in lymph node metastases than in the primary tumor. DHCR7 overexpression was also observed in tumor-bearing APC mice relative to tumor-free controls. Claudin-1 expression was found to be significantly downregulated in mice fed a high cholesterol diet compared to mice fed a low cholesterol or control diet. Finally, SW620 cells grown in cholesterol proliferated at a faster rate over 96 hours than control cells incubated in 0 μM cholesterol media, although a dose-dependent effect was not observed. Conclusions: DHCR7 is systemically overexpressed in CRC primary tumors, and expression is even further elevated in metastatic disease. Therefore, de novo cholesterol synthesis is dysregulated in tumor cells, causing a profound disruption to cholesterol homeostasis which may potentiate EMT and metastasis. High cholesterol uptake also promotes a mesenchymal phenotype in tumor cells and stimulates proliferation. Our findings indicate that dysregulation of cholesterol homeostasis increases the tumorigenicity and metastatic potential of CRC cells and may be a target for therapy. Citation Format: Blake M. Arciga, Van Nguyen, Jussuf T. Kaifi, Satyanarayana Rachagani. Dysregulated cholesterol metabolism facilitates epithelial-to-mesenchymal transition and tumor progression in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5131.

Accumulating evidence over the past decade has shown that abnormal activation of epithelial to mesenchymal transition (EMT) contributes to tumour progression and metastasis in colorectal cancer (CRC). In this study, we investigated the expression of interleukin-like EMT inducer (ILEI) and EMT-associated markers (E-cadherin, vimentin) in CRC tissues and determined the correlations between ILEI expression and clinicopathological characteristics, prognosis and EMT in CRC. In total, 194 patients diagnosed with CRC based on histopathological evaluation and those subjected to surgical resection at the First Hospital of China Medical University between 2003 and 2005 were examined. Immunohistochemical staining for ILEI, vimentin and E-cadherin was performed for each specimen. Cytoplasmic overexpression of ILEI usually accompanied down-regulation of E-cadherin and positive expression of vimentin. Conversely, ILEI was simultaneously down-regulated with overexpression of E-cadherin and negative expression of vimentin. ILEI overexpression was associated significantly with T-stage, N-stage, TNM stage and EMT phenotype (P=0.024, <0.001, <0.001 and <0.001, respectively). Multivariate analysis revealed that ILEI expression was an independent prognostic factor for patient survival. Our findings indicate that cytoplasmic ILEI expression is a potential marker of EMT and tumour progression in CRC. ILEI is an independent predictive factor associated with poor prognosis in CRC.

The Wnt/β-catenin signaling pathway plays crucial roles in tumor budding and the epithelial-mesenchymal transition (EMT). Myeloid ecotropic viral insertion site 3 (MEIS3)-a direct target of Wnt/β-catenin-promotes vagal neural crest cell migration into the gut tissue during development; however, its role in cancer progression remains unclear. In this study, the role of MEIS3 in colorectal cancer (CRC) progression was investigated. We analyzed the association between MEIS3 protein expression and the clinical stages of CRC patients, and the effect on tumor cell migration and invasion by wound healing and transwell assays. Finally, we analyzed the association between MEIS3 expression and the disease-free survival (DFS) and overall survival of CRC patients through Kaplan-Meier analysis. We found that MEIS3 expression was strongly associated with CRC progression and could be employed to assess DFS in postoperative patients. MEIS3-positive cells were mainly distributed in the growth front and tumor-stroma interface of the CRC tissues, which contain abundant EMT-active and tumor budding cells dominating cancer metastasis. Moreover, MEIS3 promoted CRC cell migration and invasion by regulating effectors including laminin subunit beta 1, matrix metalloprotein 2, and vimentin. MEIS3 protein expression increased with CRC progression according to the clinical stage, which could be used as a biomarker to stratify CRC patients. The 5-year DFS of MEIS3-high patients was poorer than that of MEIS3-low patients (40.6% vs. 61.7%; p < 0.0001). Moreover, the 5-year DFS of stage II patients with MEIS3-high expression (53.4%) was comparable to that of stage III patients with MEIS3-low expression (49.5%), while the 5-year DFS of MEIS3-high patients in stage III (30.9%) was comparable to that of stage IV patients (29.6%). This study showed that MEIS3 can promote cancer cell metastasis and thus may be a promising biomarker for higher rates of recurrence in postoperative patients with stage II/III CRC.

Colorectal cancer (CRC) continues to be a major contributor to cancer-associated death, with metastatic disease posing substantial therapeutic challenges. The epithelial-mesenchymal transition (EMT) orchestrates the transformation of polarized epithelial cells into motile mesenchymal phenotypes, characterized by enhanced migratory capacity and invasive properties. EMT is central to CRC metastasis and progression, particularly concerning its contribution to invasion, internal infiltration, and colonization. Beyond metastasis, EMT facilitates cancer cells' adaptation to diverse microenvironments, gain of stem cell-like characteristics, metabolic reprogramming, and evasion of therapeutic interventions. EMT signatures are emerging as potential prognostic biomarkers, offering valuable insights for real-time disease surveillance and personalized therapeutic strategies. Targeting EMT presents a promising therapeutic avenue to improve drug sensitivity and counteract resistance in CRC. This review systematically examines the molecular mechanisms regulating EMT in CRC, including key transcription factors; post-translational and epigenetic modifications; non-coding RNAs; and pivotal signaling pathways. Additionally, we evaluate the clinical implications of EMT in CRC progression and metastasis and critically assess emerging therapeutic strategies targeting EMT. This study lays the groundwork for developing more efficient interventions to mitigate metastasis and enhance treatment outcomes and patient survival by elucidating the intricate molecular networks that govern EMT and its contributions to CRC pathology.

Resveratrol has been an ideal alternative drug in the therapy of different cancers including colorectal cancer (CRC). Since the underlying mechanisms of resveratrol on the invasion and metastasis of CRC have not been fully elucidated, and epithelial-to-mesenchymal transition (EMT) is a key process associated with the progression of CRC, here we aimed to investigate the potential mechanism of resveratrol. We investigated the anticancer effect of resveratrol against LoVo cells in vitro and in vivo. In vivo, the impact of resveratrol on invasion and metastasis was investigated by mice tail vein injection model and mice orthotopic transplantation tumor model. In vivo imaging was applied to observe the lungs metastases, and hemaoxylin-eosin (HE) staining was used to evaluate metastatic lesions from lung metastases or hepatic metastases. In vitro, impact of resveratrol on the migration and invasion of LoVo cells was evaluated by transwell assay. Inhibition effect of resveratrol on TGF-β-induced EMT was examined by morphological observation. Epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin were detected by western blot and immunofluorescence. The promoter activity of E-cadherin was measured using a dual-luciferase assay kit. The mRNA expression of Snail and E-cadherin was measured by RT-PCR. We demonstrated that, resveratrol inhibited the lung metastases of CRC LoVo cells in vivo. In addition, resveratrol reduced the rate of lung metastases and hepatic metastases in mice orthotopic transplantation. In vitro, TGF-β1-induced EMT promoted the invasion and metastasis of CRC, reduced the expression of E-cadherin and elevated the expression of Vimentin, and activated the TGF-β1/Smads signaling pathway. But resveratrol could inhibit the invasive and migratory ability of LoVo cells in a concentration-dependent manner, increased the expression of E-cadherin, repressed the expression of Vimentin, as well as the inhibition of TGF-β1/Smads signaling pathway. Meanwhile, resveratrol reduced the level of EMT-inducing transcription factors Snail and the transcription of E-cadherin during the initiation of TGF-β1-induced EMT. Our new findings provided evidence that, resveratrol could inhibit EMT in CRC through TGF-β1/Smads signaling pathway mediated Snail/E-cadherin expression, and this might the potential mechanism of resveratrol on the inhibition of invasion and metastases in CRC. Citation Format: Qing Ji, Zhifen Han, Lihong Zhou, Hua Sui, Xuan Liu, Jianlin Ren, Fenggang Hou, Ronghua Zhao, Qi Li. Resveratrol inhibits epithelial-to-mesenchymal transition and metastasis in colorectal cancer through regulating Snail/E-cadherin expression by TGFβ1/Smads signaling pathway. [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 1689.

Purpose The epithelial–mesenchymal transition (EMT) is a key hallmark of cancer which promotes malignant progression, especially during the process of cancer invasion. A better understanding of EMT will help elucidate the molecular mechanism underlying colorectal cancer (CRC) metastasis and may provide new insights into the identification of potential biomarkers and therapeutic targets. Methods A series of bioinformatic approaches were combined and identify GLI3 as a potential key regulator in EMT. In vitro experiments were performed to knockdown GLI3 expression in two CRC cell lines and to reveal the oncogenic role of GLI3 in CRC. qRT-PCR and western blot were performed to show the influence of GLI3 in EMT and downstream pathways. The Kaplan-Meier analysis and log-rank test were used to evaluate the prognostic value of GLI3 in CRC patients. Results GLI3 was identified as a key regulator in coexpression and protein-protein interaction (PPI) networks involved in EMT. Bioinformatic analyses indicated that GLI3 had a high correlation with EMT markers in CRC. In vitro experiments showed that GLI3 knockdown attenuated the migratory and invasive capacities of CRC cells via influencing EMT property, especially by regulating phosphorylation of ERK signaling pathway. In addition, higher expression of GLI3 predicts worse prognosis in CRC patients. Conclusions In summary, we presented the first evidence that GLI3 could promote the migratory and invasive capacities of CRC cells by regulating the EMT process. Our study might provide some useful clues to a better understanding of GLI3 in EMT during CRC progression.

Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire the mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. However, as EMT is a reversible process and depends on tumor microenvironment, the precise role of EMT in cancer progression remains unclear. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancers. To evaluate the implication of EMT in the inflammation-mediated colorectal cancer progression, a live imaging system for EMT is needed on the in vitro and in vivo experiments. In this study, we generated the EMT-detectable colorectal cancer cells by introducing mesenchymal cell marker promoter-driven fluorescence protein expression vector, and investigated whether inflammation-induced EMT is detectable in vitro. Methods: To generate the EMT-detectable colorectal cancer cells HCT116-VIM635, human colorectal cancer cell line HCT116 was stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. EMT was induced in HCT116-VIM635 cells by treatment with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or by co-culture with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis. Results: Administration of IL-1β or TNF-α induced the TurboFP635 expression in consistent with morphological change like mesenchymal phenotype in HCT116-VIM635 cells. Removal of these inflammatory cytokines attenuated the TurboFP635 expression and morphological change in HCT116-VIM635 cells, suggesting the detection of reverse EMT process in these cells. Inflammatory cytokines also induced the migration and invasion properties and the expression of EMT-related markers in HCT116-VIM635 cells. Moreover, co-culture with RAW264.7 cells stimulated with LPS also induced the TurboFP635 expression and morphological change as well as inflammatory cytokines in HCT116-VIM635 cells. Conclusions: Our results suggest that this unique live imaging system for EMT has a great potential to detect reversible EMT process during inflammation-induced cancer progression. This system would be useful strategy to assess the role of EMT during inflammation-mediated cancer progression in vivo. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers. [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 1613.

Macrophages derived from circulating monocytic myeloid precursors are a major component of leukocyte infiltrate found in tumors and their role in prostate cancer progression is now emerging. Here we investigate the potential of macrophages to induce the epithelial-mesenchymal transition (EMT) in prostate cancer cells. EMT is a critical process for metastasis, and the elucidation of factors that initiate EMT would be beneficial in the development of treatments to halt the dissemination of cancer cells throughout the body. CD14+-peripheral blood monocytes were isolated from healthy donors and stimulated with either interferon gamma (INFγ) or interleukin (IL)-4, factors known to promote the differentiation of monocytes into M1 or M2-type macrophages, respectively. After 48 hours, cells from prostate cancer cell lines PC3 Luc, DU145 Luc, and ARCAP Luc were co-cultured with the macrophages for four days. The cells were passaged three times by trypsination and protein lysates were analyzed by Western Blot for the expression of EMT markers: E-cadherin and vimentin. Our results revealed that cells co-cultured with IL-4-stimulated macrophages expressed lower levels of E-cadherin and higher levels of vimentin compared to control epithelial cells. In addition, three mesenchymal-type subpopulations that were isolated from PC3 cells interacting with IL-4-treated CD14+-cells exhibited a complete loss of E-cadherin. After more than 20 passages, these cells maintained the mesenchymal characteristics in culture and showed a striking up-regulation of the transcription factor ZEB 1, whose expression has been previously correlated with Gleason grade in human prostate tumors. Concurrently, we set out to study the secreted factors by which macrophages may trigger EMT. We treated DU145 Luc and PC3 Luc cells with several cytokines found to be differentially expressed in media containing cancer cells co-cultured with IL-4-stimulated macrophages and evaluated whether any of these factors induced EMT. It was discovered that cells grown in media containing the cytokine, IL-1B, in combination with IL-4 showed a greater display of mesenchymal markers than control cells. In conclusion, these results establish that macrophages can act as potent EMT-inducers in prostate cancer cells, although the exact mediators of this function remain unknown. The data suggests that CD14+ cells stimulated with IL-4 are more effective at triggering EMT than non-stimulated or INFγ-stimulated macrophages, which may pinpoint M2-type macrophages as a mediator of EMT in vivo. Furthermore, the cytokine study revealed that macrophage-induced IL-1B in combination with IL-4 may be an important factor in the mechanism of macrophage-induced EMT. As this transition may represent a major mechanism of prostate cancer metastasis, future research will be focused on elucidating the molecules involved in order to develop novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2011-3369

Epithelial to mesenchymal transition (EMT) is a process during which cells lose their epithelial characteristics, for instance cell polarity and cell–cell contact, and gain mesenchymal properties, such as increased motility. In colorectal cancer (CRC), EMT is associated with an invasive or metastatic phenotype. In this review, we discuss recent studies exploring novel regulation mechanisms of EMT in CRC, including the identification of new CRC EMT regulators. Upregulation of inducers can promote EMT, leading to increased invasiveness and metastasis in CRC. These inducers can downregulate E-cadherin and upregulate N-cadherin and vimentin (VIM) through modulating EMT-related signaling pathways, for instance WNT/β-catenin and TGF-β, and EMT transcription factors, such as zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2. In addition, several microRNAs (miRNAs), including members of the miR-34 and miR-200 families, are found to target mRNAs of EMT-transcription factors, for example ZEB1, ZEB2, or SNAIL. Downregulation of these miRNAs is associated with distant metastasis and advanced stage tumors. Furthermore, the role of EMT in circulating tumor cells (CTCs) is also discussed. Mesenchymal markers on the surface of EMT CTCs were found to be associated with metastasis and could serve as potential biomarkers for metastasis. Altogether, these studies indicate that EMT is orchestrated by a complicated network, involving regulators of different signaling pathways. Further studies are required to understand the mechanisms underlying EMT in CRC.