Epithelial-mesenchymal Transition Related Genes Research Articles

Babao Dan inhibits the migration and invasion of gastric cancer cells by suppressing epithelial-mesenchymal transition through the TGF-β/Smad pathway.

ObjectiveTo investigate the anti-metastatic effects of Babao Dan (BBD) on gastric cancer (GC) cells (AGS and MGC80-3) and explore the underlying molecular mechanisms by which it inhibits epithelial–mesenchymal transition (EMT).MethodsAGS and MGC80-3 cells were treated with BBD. In addition, cells were treated with the EMT inducer transforming growth factor-β1 (TGF-β1). Cell viability was determined using the MTT assay, and the live cell ratio was calculated via cell counting. Cell invasion and migration were evaluated using the Transwell assay. Western blotting was performed to measure the protein expression of EMT biomarkers and related genes.ResultsBBD inhibited the viability, migration, and invasion of AGS and MGC80-3 cells, but it did not reduce the live cell ratio. Furthermore, BBD inhibited the expression of N-cadherin, vimentin, zinc finger E-box binding homeobox (ZEB)1, ZEB2, Twist1, matrix metalloproteinase (MMP)2, MMP9, TGF-β1, and p-Smad2/3, whereas E-cadherin expression was increased in AGS and MGC80-3 cells to different degrees. Using a GC cell model of EMT induced by TGF-β1, we proved that BBD inhibited p-Smad2/3 and N-cadherin expression, cell migration, and cell invasion.ConclusionBBD suppressed cell migration and invasion by inhibiting TGF-β–induced EMT and inactivating TGF-β/Smad signaling in GC cells.

NGF from pancreatic stellate cells induces pancreatic cancer proliferation and invasion by PI3K/AKT/GSK signal pathway.

Pancreatic cancer (PC) is a continuously high lethal disease, and the tumour microenvironment plays a pivotal role during PC progression. Herein, we focus on that the Nerve growth factor (NGF)/Tropomyosin‐related kinase A (TrkA), in pancreatic stellate cells‐pancreatic cancer cells (PSCs‐PC cells) co‐culture system, influences PC proliferation and invasion. The model of PC cells and PSCs was directly co‐cultured in a no‐touch manner, using the Transwell as the co‐culture system. NGF and TrkA expression was measured in cultured system by real‐time PCR, immunofluorescence, Western blotting analysis or ELISA. Small interfering RNA transfection was used to regulate the expression of TrkA in PC cells. The promotion of cancer invasion was investigated using Matrigel Transwell assay. In our study, NGF/TrkA is overexpressed in PSCs‐PC cells co‐culture system and promotes the invasion and proliferation of PC cells. And the epithelial‐mesenchymal transition‐related genes are influenced by si‐TrkA. What's more, NGF/TrkA regulates the PC cell proliferation and invasion via activation of PI3K/AKT/GSK signalling. The present study demonstrated NGF/TrkA promoted the PC cell proliferation and invasion in the co‐culture system by the activation of the PI3K/AKT/GSK signal cascade, providing a potential therapeutic target for PC patients.

Increased Expression of LIPC Is Associated with Aggressive Phenotype of Borrmann Type 4 Gastric Cancer

PurposeTo investigate lipase C hepatic type (LIPC) expression in Borrmann type 4 gastric cancer and its correlation with clinical outcome. The biological roles of LIPC in Borrmann type 4 gastric cancer progression were also investigated. MethodsWe determined LIPC expression in 324 primary gastric cancer tissues and 178 matched adjacent non-tumor tissues by immunohistochemistry. We explored the role of LIPC in Borrmann type 4 gastric cancer cell (OCUM-1) migration, invasion, proliferation, cell cycle, and expression of epithelial-mesenchymal transition-related genes by knocking down LIPC expression. ResultsLIPC expression was upregulated in Borrmann type 4 gastric cancer tissues compared with other types of gastric cancer and adjacent non-tumor tissues. High LIPC expression correlated with lymph node metastasis, advanced TNM stage, and poor overall survival in Borrmann type 4 gastric cancer patients. Multivariate analysis demonstrated that high LIPC expression was an independent prognostic factor in patients with Borrmann type 4 gastric cancer. By reducing LIPC expression, OCUM-1 cell invasion and migration were suppressed and Snail and MMP2 expression was downregulated, while E-cadherin expression was upregulated. ConclusionsHigh LIPC expression correlates with poor clinical outcome and plays an important role in regulating cell migration and invasion in Borrmann type 4 gastric cancer.

Effect of epigenetic modulation on cancer sphere

Background : Cancer stem cell properties are highly relevant to the biology of treatment-resistant cancers. Epigenetic modification regulates gene expressions by chromatin remodeling during malignant transformation. The aim of this study was to elucidate the possible strategy for cancer stem cells focusing on epigenetic modification. Methods : We made cancer sphere from HepG2 cells, and we added Histone deacetylase (HDAC) inhibitor, valproic acid to cancer sphere. And we compared methylation status and the gene expression between normal HepG2 and cancer sphere groups, and between cancer sphere and sphere with HDAC inhibitor treatment groups. Results : Valproic acid (VPA) cancelled this spheroid formation. In comparison between normal HepG2 and cancer sphere, the number of methylation status changes more than 0.1 of beta level was 826 probes, and we could isolate some epithelial-mesenchymal transition (EMT) related genes. And VPA reduced the expressions of EMT related genes in sphere with RT-PCR. On the other hand, in comparison between cancer sphere and sphere with VPA treatment, we detected 29 probe of methylation status change, and VPA reduced the expressions of Bcl-6 in sphere. Conclusions : HDAC inhibitor affected the methylation status of cancer stem cells. Histone-acetylation might overcome treatmet-resistant cancer through the regulation of cancer stem cell. J. Med. Invest. 67 : 70-74, February, 2020.

Cinobufacini Inhibits Colon Cancer Invasion and Metastasis via Suppressing Wnt/β-Catenin Signaling Pathway and EMT.

Cinobufacini is a well-known Chinese medicine extracted from Venenum Bufonis, also called Chan Su. It has been used clinically for various cancers, including colon cancer. However, the function of Cinobufacini on colon cancer invasion and metastasis, and its underlying molecular mechanism, is still not clear. In this study, we investigated the function and mechanism of Cinobufacini on colon cancer invasion and metastasis both in vitro and in vivo studies. Human colon cancer cells were cultured. CCK assay was used to detect the effect of Cinobufacini on colon cancer cells proliferation. The invasion and migration abilities were observed by transwell assays, and the expression of invasion and migration related genes MMP2, MMP9, and epithelial-to-mesenchymal transition (EMT) relate genes were observed by Western blot assays. An orthotopic xenograft model in nude mice was established using colon cancer HCT116 cells, and the function of Cinobufacini on colon cancer invasion and metastasis were observed in vivo. We found Cinobufacini significantly inhibited colon cancer cell proliferation in a dose/time-dependent manner; the invasion and migration abilities of colon cancer were decreased after treated with Cinobufacini. The metastasis and EMT related genes MMP9, MMP2, N-cadherin and Snail were obviously down-regulated, while the expression of E-cadherin was up-regulated after treatment with Cinobufacini. The Wnt/-catenin signaling pathway related genes were observed using WB,and results show that the expression of -catenin, wnt3a, c-myc, cyclin D1, and MMP7 were all down-regulated after being treated with cinobufacini, while the expression of APC was up-regulated. In vivo studies of the volume and weight of orthotopic xenograft tumors showed significantly shrinkage in the Cinobufacini group compared to the control group. The enterocoelia and liver metastasis tumors were significantly decreased, and the expression of MMP9, MMP2, and -catenin were also down-regulated, while E-cadherin was up-regulated in vivo after the treatment with Cinobufacini. Our data proves that Cinobufacini can inhibit colon cancer invasion and metastasis both in vitro and in vivo; the mechanism is related by suppressing the Wnt/-catenin signaling pathway and then inhibiting the EMT of CRC.

Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment

Tumor cells interact with the microenvironment that specifically supports and promotes tumor development. Key components in the tumor environment have been linked to various aggressive cancer features and can further influence the presence of subpopulations of cancer cells with specific functions, including cancer stem cells and migratory cells. To model and further understand the influence of specific microenvironments we have developed an experimental platform using cell-free patient-derived scaffolds (PDSs) from primary breast cancers infiltrated with standardized breast cancer cell lines. This PDS culture system induced a series of orchestrated changes in differentiation, epithelial-mesenchymal transition, stemness and proliferation of the cancer cell population, where an increased cancer stem cell pool was confirmed using functional assays. Furthermore, global gene expression profiling showed that PDS cultures were similar to xenograft cultures. Mass spectrometry analyses of cell-free PDSs identified subgroups based on their protein composition that were linked to clinical properties, including tumor grade. Finally, we observed that an induction of epithelial-mesenchymal transition-related genes in cancer cells growing on the PDSs were significantly associated with clinical disease recurrences in breast cancer patients. Patient-derived scaffolds thus mimics in vivo-like growth conditions and uncovers unique information about the malignancy-inducing properties of tumor microenvironment.

Multi-omics analysis reveals epithelial-mesenchymal transition-related gene FOXM1 as a novel prognostic biomarker in clear cell renal carcinoma.

Identification of novel clinical biomarker in clear cell renal carcinoma (ccRCC) is warranted. Integrating transcriptome (n=1669), DNA methylation (n=577) and copy number data (n=832), we developed a method to identify driver biomarkers by analyzing the omics-level dynamics of Epithelial-Mesenchymal Transition (EMT)-related genes in ccRCC. We first identified 504 expression dynamic changed genes involved in ccRCC-associated key pathways such as EMT, cell cycle, EGFR and PI3K/AKT signaling. Further analysis identified 229 (90 gene promoters) aberrant expression quantitative trait methylation (eQTM) and 256 genes with expression quantitative trait copy number (eQTCN) alterations. Among them, FOXM1 was affected by both eQTM and eQTCN. FOXM1 copy number amplification (115/500, 23% of patients), occurred in an amplified peak in chromosome 12q13.3, was enriched in late-stage ccRCC samples and was associated with worse survival. FOXM1-overexpressed pT3 patients with distant metastasis showed ~25% shorter overall survival in both training (log-rank P=0.006) and validation (log-rank P=0.018) cohorts. The eQTM-gene hybrid signature (cg00044170 and FOXM1), superior to either gene expression or DNA methylation alone, showed great potential in diagnosing localized ccRCC in training (area under curve = 0.958) and validation datasets. FOXM1 could be a novel prognostic biomarker and shed light for early diagnosis at molecular level in ccRCC.

Mechanism of aberrant long non-coding RNA expression in an adriamycin-resistant liver cancer cell strain

Background and aimsAberrant long non-coding RNA (lncRNA) expression in cancer can be used as a potential diagnostic biomarker and therapeutic target. In the present study we determined the potential pathogenic mechanism underlying significant aberrant expression of lncRNAs in HepG2-ADR. MethodsFirst, we identified different levels of lncRNA expression in liver cancer and adjacent non-tumor tissues obtained from public data (GSE70880) in NCBI. Then, the results were verified in a sensitive liver cancer cell line (HepG2) and a adriamycin-resistant liver cancer cell line (HepG2-ADR). Then, the effects of lncRNAs on the phenotype and some biological characteristics were also determined in HepG2 and HepG2-ADR through overexpression and using siRNA interference methods. ResultsWe showed that lncRNA ENST00000425005 is highly expressed in a liver cancer-resistant cell line when compared with adjacent non-tumor tissues based on bioinformatics analysis and qPCR verification. Compared with the control group, overexpression of lncRNA ENST00000425005 significantly promoted proliferation and adhesiveness, but inhibited apoptosis in HepG2-ADR cells. In contrast, interference of lncRNA in HepG2-ADR cells suppressed proliferation and adhesiveness, and induced apoptosis. ConclusionIn conclusion, lncRNA ENST00000425005 promotes cell proliferation and invasion in drug-resistant liver cancer cells by regulating epithelial–mesenchymal transition-related gene expression and participating in the regulation of EGF and FGF7.

Homeobox B8 Targets Sterile Alpha Motif Domain-Containing Protein 9 and Drives Glioma Progression.

Gliomas are the most commonly occurring tumors of the central nervous system. Glioblastoma multiforme (GBM) is the most malignant and aggressive brain cancer in adults. Further understanding of the mechanisms underlying the aggressive nature of GBM is urgently needed. Here we identified homeobox B8 (HOXB8), a member of the homeobox family, as a crucial contributor to the aggressiveness of GBM. Data mining of publicly accessible RNA sequence datasets and our patient cohorts confirmed a higher expression of HOXB8 in the tumor tissue of GBM patients, and a strong positive correlation between the expression level and pathological grading of tumors and a negative correlation between the expression level and the overall survival rate. We next showed that HOXB8 promotes the proliferation and migration of glioblastoma cells and is crucial for the activation of the PI3K/AKT pathway and expression of epithelial-mesenchymal transition-related genes, possibly through direct binding to the promoter of SAMD9 (Sterile Alpha Motif Domain-Containing Protein 9) and activating its transcription. Collectively, we identified HOXB8 as a critical contributor to the aggressiveness of GBM, which provides insights into a potential therapeutic target for GBM and opens new avenues for improving its treatment outcome.

TRIP13 interference inhibits the proliferation and metastasis of thyroid cancer cells through regulating TTC5/p53 pathway and epithelial-mesenchymal transition related genes expression

This study aimed to investigate the effect of interfering thyroid hormone receptor interacting protein 13 (TRIP13) expression on the proliferation, apoptosis and metastasis of thyroid cancer (TC) cells and the involved mechanisms. RT-PCR, immunohistochemical analysis and western blot found that compared with normal tissues, the expressions of TRIP13 and N-cadherin in TC tissues were significantly increased, while the expressions of tetratricopeptide repeat protein 5 (TTC5), p-p53 and E-cadherin were significantly decreased (P < 0.05). RT-PCR and western blot revealed that compared with C643 cells, TRIP13 expression in TPC1 cells and BHT101 cells increased significantly (P < 0.05). Therefore, TPC1 cells and BHT101 cells were selected for subsequent experiments. Interference efficiency of TRIP13 interference sequences (sh-TRIP13) was verified by RT-PCR and western blot. After sh-TRIP13 transfection, cell viability and migration rates of cells at 24 h and 48 h decreased significantly (P < 0.05); the number of S phase cells increased remarkably, while the number of G1 and G2 phase cells decreased significantly (P < 0.05); cell apoptosis was enhanced sharply (P < 0.05); cell numbers in the lower chamber of Transwell assay were reduced significantly (P < 0.05). RT-PCR and western blot found that compared with the Control group, expressions of TTC5 and E-cadherin in sh-TRIP13 group were elevated sharply, while N-cadherin expression decreased significantly (P < 0.05). Compared with the Control group, sh-TRIP13 transfection elevated the ratio of p-p53 expression to p53 expression (p-p53/p53) remarkably (P < 0.05). In conclusion, TRIP13 interference inhibited the proliferation and metastasis of thyroid cancer cells through regulating TTC5/p53 pathway and epithelial-mesenchymal transition related genes expression.

Prognostic Analysis of Hepatocellular Carcinoma With Hepatitis C Virus Infection Using Epithelial-Mesenchymal Transition Gene Profiles

BackgroundEpithelial-mesenchymal transition genes have prognostic influence on hepatocellular carcinoma (HCC). Previously, the following four epithelial-mesenchymal transition–related genes were considered to be significantly influential: E-cadherin (CDH1), inhibitor of DNA binding 2 (ID2), matrix metalloproteinase 9 (MMP9), and transcription factor 3 (TCF3). A prognostic prediction model, NRISK4 = (−0.333 × [CDH1] − 0.400 × [ID2] + 0.339 × [MMP9] + 0.387 × [TCF3]) was constructed, but from patients with HCC with predominantly hepatitis B virus infection. We therefore aim to validate if this model also fits patients with HCC and hepatitis C virus (HCV) infection. MethodsWe collected HCC tissue samples from 67 patients with HCV infection. Discrimination of the NRISK4 was re-estimated using receiver operating curve analysis and we redefined the appropriate cutoff value. Using this cutoff value, patients were divided into two groups (high/low risk patients) and we compared their clinicopathological factors and prognosis. ResultsArea under the curve of NRISK4 prediction was 0.70 and an appropriate cutoff value was 3.19 in this cohort. Patients were divided into high- (n = 25) and low-risk (n = 42) patients for prognosis. There were no significant differences in tumor factors between the two groups. Cancer-specific survival rates at 5 y after surgery on high- and low-risk patients were 45% and 68%, respectively (P = 0.02). At 2 y after surgery, recurrence rates were 68% and 37% among high- and low-risk patients, respectively (P = 0.01). Aggressive recurrences were highly observed in the high-risk patients (P = 0.01). ConclusionsNRISK4 model could also successfully validate prognosis of patients with HCC with HCV infection similarly to in the previous report of patients with hepatitis B virus infection, especially in the early period after surgery.

Role of TGF‑β in the motility of ShcD‑overexpressing 293 cells.

The newly identified Src homology and collagen (Shc) family member ShcD was observed to be upregulated in 50% of vertical growth phase and metastatic melanomas. The aim of the present study was to investigate the mechanism by which ShcD mediates cell motility. 293 cell lines were altered to stably express GFP (GF) or GFP-ShcD (G5). Treatment of the cells with transforming growth factor (TGF)β2 promoted extracellular signal-regulated kinase (ERK) phosphorylation and, to a lesser extent, Smad2 phosphorylation in GFP-ShcD-expressing cells but not in GFP-overexpressing cells. GFP-ShcD-expressing cells exhibited upregulated expression of certain epithelial-mesenchymal transition-related genes, such as snail family transcriptional repressor 1 and SLUG, than GFP-expressing cells. Higher levels of ERK were found in the nuclear fraction of GFP-ShcD-expressing cells than that of GFP-expressing cells. Overall, GFP-ShcD-expressing cells demonstrated enhanced migration compared with GFP-expressing cells. A slight increase in cell migration was observed in both cell lines (GF and G5) when the cells were allowed to migrate towards conditioned medium derived from TGFβ2-treated GFP-ShcD expressing cells. Collectively, ShcD upregulation was proposed to induce cell migration by affecting the expression of certain epithelial-mesenchymal transition-related genes. Thus, our findings may improve understanding of the role of ShcD in cell migration.

Abstract 1031: Single-cell analysis reveals a possibility of epithelial to circulating tumor cell transition (ECT) when tumor cells enter into the bloodstream

Abstract Tumor cells face the enviormental changes when leave tumor tissue enter into bloodstream. The environmental changes stimulate tumor cells to adapt the new enviorments with cell functional changes. Epithelial to mesenchymal transition (EMT) is well known and it explains the changes relatively in a portion of the circuating tumor cells (CTC) and in the relative later stage. We use the single-cell molecular analysis on house keeping genes, apoptosis genes, metabolism genes, Epithelial genes, EMT genes, metastasis genes, stem cell genes, and proliferation genes. The results reveal that less than half of individual CTCs express normal level of house keeping genes; apoptosis genes are not expressed at the normal level (reduced or not detectable) in the CTCs without house keeping genes expression; the expression of metabolism genes are relatively low especially in the CTCs without GAPDH expression; the expression level of Epithelial genes are low and the EpCAM and KRT7 are undetectable in most CTCs, but KRT8/18/19 are remaining high expression in the CTCs with GAPDH expressed; EMT genes of VIM, Twist1, MMP-2, TGFB1 are highly expressed in CTCs with GAPDH expressed but no or low expression in the CTCs without GAPDH expression; the expression of metastasis genes are relatively high in the CTCs with GAPDH expressed, but not in the CTCs without the expression of GAPDH; the expression of CD44, CD24, Bmi1, Oct-4, CXCR4, CD9, CXCL2, MAPK14 are highly expressed in the CTCs with GAPDH expressed but not in the CTCs without GAPDH expression; CD44 expressed in some CTCs with higher level than CD24, but much more individual CTCs (with GAPDH expressed) showed higher expression of CD24 than that of CD44 per single-cell level; the expression of proliferation genes are reduced in the CTCs with GAPDH expressed and not detectable in the CTCs without GAPDH expression. Based on the results, we hypthesize "Epithelial to circulating transition (ECT)" at the early stage after the tumor cells enter into bloodstream and followed by "CMT (circulating to mesenchymal transition and then MEC (mesenchymal to epithelial transition)". The ECT may includes the cell changes related to surviving, metabolism level, remaining the characteristics of metastatsis, stem cell signatures, Epithelial related genes and EMT related genes, proliferation genes and others. Studying the early changes of tumor cells enter into bloodstream maybe very improtant for gaining the understanding of the characteristics of survival cells and metastatic tumor cells and also important for basic research and clinical applications. Note: This abstract was not presented at the meeting. Citation Format: Glenn Deng, Stefanie Jeffrey. Single-cell analysis reveals a possibility of epithelial to circulating tumor cell transition (ECT) when tumor cells enter into the bloodstream [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1031.

Abstract 1041: Casein kinase 2 modulates epithelial mesenchymal transition through Helicobacter pylori CagA dependent pathway in gastric cancer cells

Abstract Helicobacter pylori is the most important carcinogen in human gastric cancer. However, the pathogenic molecular mechanism which underlies the carcinogenesis in gastric cancer by H. pylori remains largely unknown. In this study, we understand that molecular biological mechanism related to gastric carcinogenesis by investigating the role of CK2 in EMT. Casein kinase 2 is a serine/threonine protein kinase and consists of two catalytic subunits (α or α’) and regulatory subunits (β). CK2 regulates many substrates and its involved in cell growth, proliferation, survival, angiogenesis, invasion. Epithelial-to-mesenchymal transition (EMT) is involved in many signaling pathways, but the key regulatory kinases in this process have not been clearly identified. In our previous study, we reported the cytotoxin-associated gene A (CagA) protein of H. pylori induces cell migration and invasion through increased CK2α activity in gastric epithelial cells. Although the role of CK2 catalytic subunits has remained largely uncharacterized, several studies have recently focused on regulator subunits in EMT. Here, we analyzed the expression level of CK2α did not altered, whereas CK2β decreased in CagA-dependent pathway. Moreover, expression of ectopic CK2β was downregulated by CagA. This suggests that CagA negatively regulates the stability of CK2β protein. We examined the level of ubiquitinated CK2β were higher in HP60190 infected cells than in control cells. Thus, CK2β is degraded by the proteasomal machinery following CagA of H. pylori. In addition, CagA binds both CK2α and CK2β, which results in suppression of CK2β binding by infected HP60190, but does not suppress CK2α binding. Furthermore, downregulation of CK2β increased Snail such as CK2 target genes, EMT-related marker in H. pylori-infected gastric cancer cells. Therefore, the role of Snail regulated by CK2β should be considered when developing cancer therapeutic. Overall, CK2 tetramer subunits might control the function of CagA and EMT related genes, thereby regulating CagA-dependent pathology. Taken together, the results of the present study suggest that the CK2 regulatory subunit has diverse effect on CagA-dependent cellular processes. A pharmacological activator or activator of CK2β may have potential as a therapeutic agent for gastric cancer associated with CagA. Citation Format: SoDam Lee, Bo Ram Hwang, Jung Chang Yun, Byeong Min Yu, Yong Chan Lee. Casein kinase 2 modulates epithelial mesenchymal transition through Helicobacter pylori CagA dependent pathway in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1041.

Abstract 275: A possible relationship between gene mutation profiles and activity of E7386 in in vitro models of colorectal cancer organoids

Abstract A novel beta-catenin/CBP binding modulator E7386 is expected to affect colorectal carcinoma (CRC) cells particularly those with aberrant activation of Wnt/beta-catenin signaling pathway. The Wnt signaling pathway is recently reported to play important roles for activation of not only CRC cells but also cancer-associated fibroblasts (CAFs). Here, we established an in vitro model of CRC-derived organoids as well as a co-culture method of the CRC organoids with CAFs, and effects of E7386 on the growth of the organoids were examined. Fragments of CRC tissues from a total of 45 patients who underwent surgery were obtained, dissociated into single cells, which were seeded on polymerized Matrigel (Corning), and cultured with serum-free media containing EGF and Noggin optimized for organoids using 12-well plates. Fourteen CRC organoids from 45 cases were successfully established. For 2D culture of CAFs from CRC tissues of each patient, RPMI medium containing 10% FBS was used. A co-culture method of the CRC organoids with CAFs was applied using a double chamber system with transwell inserts in 24-well plates. Cell viabilities of the organoids and CAFs were measured using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega). Gene aberrations in original CRC tissues were analyzed using NCC Oncopanel, and gene expression profiles in established CRC organoids and CAFs were analyzed using SurePrint G3 Human GE microarray (Agilent). [Effects of CAFs on cell viabilities and gene expressions of organoids] Cell viabilities of organoids and CAFs appeared to increase at 72 hours after beginning of the culture by 2 to 8 and 1 to 2-fold, respectively. When organoids were co-cultured with CAFs, cell viabilities of organoids were increased and epithelial-mesenchymal transition-related gene expressions were dramatically changed in several cases, but not in all cases. The organoids co-cultured with CAFs appeared to be more sensitive to E7386 as compared to those cultured alone. [Possible relationship between gene mutations in CRCs and activity of E7386 in CRC organoids] Cell viabilities of organoids without APC mutations (n=1) and organoids with APC and TP53 mutations alone (n=2) were less than those of organoids with other oncogene mutations, e.g., KRAS or PIK3CA (n=4). Efficacy of E7386 on the cell viabilities of organoids with APC and TP53 mutations alone was found at lower concentrations at 30-100 nM, as compared to those of organoids without APC mutations. From the results, CRC organoids were established from 14 patients, and activity of E7386 appeared to be related to gene mutation profiles in the established CRC organoids. Citation Format: Toshio Imai, Mie Naruse, Masako Ochiai, Yoichi Ozawa, Takashi Owa, Atsushi Ochiai. A possible relationship between gene mutation profiles and activity of E7386 in in vitro models of colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 275.

Quasimesenchymal phenotype predicts systemic metastasis in pancreatic ductal adenocarcinoma

Metastasis following surgical resection is a leading cause of mortality in pancreatic ductal adenocarcinoma. Epithelial-mesenchymal transition is thought to play an important role in metastasis, although its clinical relevance in metastasis remains uncertain. We evaluated a panel of RNA in-situ hybridization probes for epithelial-mesenchymal transition-related genes expressed in circulating tumor cells. We assessed the predictive value of this panel for metastasis in pancreatic ductal adenocarcinoma and, to determine if the phenotype is generalizable between cancers, in colonic adenocarcinoma. One hundred fifty-eight pancreatic ductal adenocarcinomas and 205 colonic adenocarcinomas were classified as epithelial or quasimesenchymal phenotype using dual colorimetric RNA-in-situ hybridization. SMAD4 expression on pancreatic ductal adenocarcinomas was assessed by immunohistochemistry. Pancreatic ductal adenocarcinomas with quasimesenchymal phenotype had a significantly shorter disease-specific survival (P = 0.031) and metastasis-free survival (P = 0.0001) than those with an epithelial phenotype. Pancreatic ductal adenocarcinomas with SMAD4 loss also had lower disease-specific survival (P = 0.041) and metastasis-free survival (P = 0.001) than those with intact SMAD4. However, the quasimesenchymal phenotype proved a more robust predictor of metastases—area under the curve for quasimesenchymal = 0.8; SMAD4 = 0.6. The quasimesenchymal phenotype also predicted metastasis-free survival (P = 0.004) in colonic adenocarcinoma. Epithelial-mesenchymal transition defined two phenotypes with distinct metastatic capabilities—epithelial phenotype tumors with predominantly organ-confined disease and quasimesenchymal phenotype with high risk of metastatic disease in two epithelial malignancies. Collectively, this work validates the relevance of epithelial-mesenchymal transition in human gastrointestinal tumors.

CXCR4 regulates the development of bladder cancer via wnt/β-catenin signaling pathway

Objective To investigate the role of chemokine receptor 4 (CXCR4) in the development of bladder cancer. Methods QPCR was used to detect the expression of CXCR4 mRNA in normal bladder tissues, bladder cancer tissues and bladder cancer cell lines SW780, 5637 and T24. MTT, colony formation, and transwell migration and invasion assays were performed in SW780 cells after treatment with the CXCR4 antagonist AMD3465. β-catenin, Wnt/β-catenin pathway targeted genes and epithelial-mesenchymal transition (EMT) related genes were detected by Western blot. The effect of AMD3465 on the growth of human bladder cancer SW780 cell xenografts was evaluated using a nude mice model. Results The expression of CXCR4 in bladder cancer and bladder cancer cell lines was significantly higher than that in normal bladder tissues. AMD3465 could effectively inhibit the proliferation, colony formation, migration, and invasion of SW780 cells, meanwhile, it significantly inhibited the expression of β-catenin and Wnt/β-catenin pathway targeted genes. The expression of EMT related gene E-cadherin was significantly up-regulated. Conclusions CXCR4 affects the development of bladder cancer by regulating Wnt/β-catenin signaling pathway. Key words: Urinary Bladder Neoplasms; Receptors, CXCR4; Cell Proliferation; Cell Movement

Highlights of This Issue

A metalloendopeptidase, nardilysin (NRDC), has been suggested to play important roles in inflammation and several cancer types. Yoh and colleagues explored the feasibility of serum NRDC as a prognostic biomarker in patients undergoing surgical resection for intrahepatic cholangiocarcinoma (ICC), the second-most common primary liver cancer with high metastatic rate. As a result, high NRDC levels were associated with poorer prognosis. Serum NRDC levels also were correlated with epithelial mesenchymal transition-related genes in primary tumors. Together, serum NRDC can be a clinically relevant tool for predicting the postoperative outcomes of ICC and therefore contribute to develop a treatment strategy.Up to 50,000 thyroidectomies are performed unnecessarily in the United States annually due to the inability to diagnose benign versus cancerous thyroid nodules. By using genome wide DNA methylation analysis of 109 thyroid tissues, Yim and colleagues found that benign nodules differ from adjacent thyroid tissues and malignant nodules. By using tissue specific epigenetic patterns for benign and malignant thyroid nodules, the authors developed a highly accurate thyroid cancer diagnostic test with an estimated 97% PPV and 100% NPV. Thus, a tissue-specific DNA methylation approach has the potential to provide standard-of-care cancer diagnoses for thyroid nodules.The cellular adhesion molecule VCAM-1 is upregulated on vascular endothelial cells in disease. Using mouse models of brain metastases, Cheng and colleagues show that vascular VCAM-1 is expressed early following metastatic seeding in the brain across a number of different tumor types. Moreover, an MR imaging probe targeted to VCAM-1 enabled detection of micrometastatic deposits that were undetectable using the standard clinical diagnostic method of gadolinium-enhanced MRI. This VCAM-1-targeted MRI approach has the potential to drastically alter the management of patients at risk of brain metastasis in existing treatment paradigms and will lead to greater personalization of care.SGK1 is a conserved member of the AG family of serine/threonine kinases that is highly expressed at the tops of intestinal crypts, suggesting a role in differentiation. SGK1 expression is often reduced in colorectal cancer, with poorly differentiated tumors resulting in a worse prognosis. To explore its role in differentiation, Lee and colleagues conducted a multiomics approach to demonstrate that SGK1 re-expression directly promotes the differentiation of colorectal cancer cells, while inhibiting metastatic potential in a mouse orthotopic xenograft model. This work proposes a greater focus upon therapeutic agents that promote tumor differentiation, rather than simply targeting cell survival, for future research.

Cancer IgG, a potential prognostic marker, promotes colorectal cancer progression.

ObjectiveCurrently, no satisfactory targets for colorectal cancer or markers for immunotherapy and diagnosis and prognosis are available. Immunoglobulin G (IgG) is widely expressed in many cancers, and it promotes cancer progression. This study explored the role of cancer-derived IgG (CIgG) in colorectal cancer.MethodsFirst, using a monoclonal antibody to CIgG, we examined the expression levels of CIgG in colorectal cancer cell lines by western blot and immunofluorescence analyses and in tissue specimens by immunohistochemistry. Second, the variable region gene was amplified by nested polymerase chain reaction (PCR), and PCR products were sequenced and analyzed. Third, we investigated the effect of CIgG on colorectal cancer cells by cell proliferation, wound healing, migration and invasion assays, and colony formation assay. Fourth, we performed in vivo tumorigenicity experiments to explore the effect of CIgG on tumorigenicity. Finally, we used RNA-seq analysis and co-immunoprecipitation experiments to further clarify possible mechanisms of CIgG. ResultsWe found that CIgG is widely expressed in colorectal cancer cells, and the overexpression of CIgG indicates significantly poor colorectal cancer prognosis. Furthermore, CIgG knockdown significantly inhibits the proliferation, migration and invasion ability of cells, and tumor growth in vivo. RNA-seq analysis indicated that CIgG knockdown results primarily in changes in expression of apical junction and epithelial-mesenchymal transition-related genes. CIgG may be involved in colorectal cancer invasion and metastasis through interacting with E-cadherin. ConclusionsCIgG is a potential human oncogene in colorectal cancer and that it has potential for application as a novel target in targeted therapy and a marker for prognostic evaluation.

Ginsenoside Rd Inhibits the Metastasis of Colorectal Cancer via Epidermal Growth Factor Receptor Signaling Axis.

Ginsenoside Rd is a saponin from ginseng and has been reported to have various biological activities. However, the effect of ginsenoside Rd on the metastasis of colorectal cancer (CRC) remains unknown. Here, we found that ginsenoside Rd decreased the colony-forming ability, migration, invasion, and wound-healing abilities of CRC cells, although it did not affect cell proliferation. In addition, using an inverse-docking assay, we found that ginsenoside Rd bound to epidermal growth factor receptor (EGFR) with a high binding affinity, inducing the downregulation of stemness- and epithelial-mesenchymal transition-related genes; these were partially rescued by either exogenous EGF treatment or ectopic expression of SOX2. Furthermore, ginsenoside Rd significantly decreased the number and size of tumor metastasis nodules in the livers, lungs, and kidneys of mouse model of metastasis. © 2018 IUBMB Life, 71(5):601-610, 2019.