Expression Of EMT-related Genes Research Articles

Upregulation of Mir-21 Levels in the Vitreous Humor Is Associated with Development of Proliferative Vitreoretinal Disease.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by post-transcriptional inhibition of mRNA translation. Dysregulation of miRNAs, including circulating miRNAs, has been reported to play an important role in the development of various diseases, including fibrotic diseases. Aberrant expression of miRNAs in the vitreous humor of vitreoretinal diseased eyes has been reported. However, the expression pattern of miRNAs present in the vitreous humor of proliferative vitreoretinal disease (PVD) patients, including proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), remains unknown. To investigate the factors important for the development of PVD, we characterized the miRNAs present in the vitreous humor of PVD patients and analyzed the expression profiles of 377 miRNAs using quantitative polymerase chain reaction-based miRNA arrays. The expression of a specific subset of miRNAs, previously reported to be associated with the development of angiogenesis and fibrosis, was significantly altered in the vitreous of PVD patients. Among these miRNAs, we identified miR-21 as a candidate fibrotic miRNA with an important role in the pathogenesis of PVD. Increased miR-21 levels in the vitreous were associated with retinal fibrosis, including PVR and PDR. Because epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPECs) plays a critical role in retinal fibrosis, the expression of miR-21 in human RPECs was determined. Its expression in RPECs was induced by transforming growth factor-β, a key growth factor involved in fibrogenesis, and was enhanced by high glucose culture conditions, suggesting that miR-21 expression positively correlates with disease progression. Gain- and loss-of-function studies revealed that miR-21 promoted cell proliferation and migration of ARPE-19 cells without affecting EMT-related gene expression. Together, our studies have identified miR-21 as a potential disease-modifying miRNA in the vitreous humor that is involved in the development of retinal fibrosis and may be a novel marker of PVD.

Angiotensin II promotes endometrial cancer cell survival.

Endometrial cancer(EC) is one of the most common female cancers. One of the key processes involved in EC development is uncontrolled proliferation stimulated by local factors such as angiotensin. The aim of the present study was to evaluate the influence of angiotensinII(AngII) on human EC cells. Biological assays and gene expression analysis were performed on three cell lines: ISH, MFE-296 and MFE-280. Our results indicated that at the beginning of cancerogenesis AngII induced abnormal proliferation at lower doses. We also showed that dose-dependent induction of proliferation was connected with changes in the expression of MKI67, CCND1 and CCNE1 genes in well- and poorly differentiated cancer cells. After AngII treatment, poorly differentiated endometrial cancer cell line acquired a mesenchymal phenotype, which was characterized by induced expression of EMT-related genes (VIM, CD44, SNAI1, ZEB1 and ZEB2). Our study revealed that AngII influences EC cells in terms of cancer-related processes, and is responsible for increased proliferation, reduction in apoptosis, increased mobility and modulation of adhesion potential. Its effect and effectiveness appear to be highly connected with the differentiation status of the cancerous cells, as AngII appears to play a crucial role in the early and late stages of malignant transformation.

Effect of benzophenone-1 and octylphenol on the regulation of epithelial-mesenchymal transition via an estrogen receptor-dependent pathway in estrogen receptor expressing ovarian cancer cells

Epithelial-mesenchymal transition (EMT) is an important process in embryonic development and cancer progression and metastasis. EMT is influenced by 17β-estradiol (E2), an endogenous estrogen. Benzophenone-1 (2,4-dihydroxybenzophenone, BP-1) and 4-tert-octylphenol (OP) are suspected endocrine disrupting chemicals (EDCs) because they can exhibit estrogenic properties. In this study, we examined whether BP-1 and OP can lead to EMT of BG-1 ovarian cancer cells expressing estrogen receptors (ERs). A wound healing assay and western blot assay were conducted to show the effect of BP-1 and OP on the migration of BG-1 cells and protein expression of EMT-related genes. BP-1 (10−6 M) and OP (10−6 M) significantly enhanced the migration capability of BG-1 cells by reducing the wounded area in the cell monolayer relative to the control, similar to E2 (10−9 M). However, when BG-1 cells were co-treated with ICI 182,780, an ER antagonist, the uncovered area was maintained at the level of the control. N-cadherin, snail, and slug were increased by BP-1 and OP while E-cadherin was reduced compared to the control. However, this effect was also restored by co-treatment with ICI 182,780. Taken together, these results indicate that BP-1 and OP, the potential EDCs, may have the ability to induce ovarian cancer metastasis via regulation of the expression of EMT markers and migration of ER-expressing BG-1 ovarian cancer cells.

Extracellular High mobility group box 1-induced epithelial mesenchymal transition through β-catenin pathway in human airway epithelial cells

Abstract High mobility group box 1 (HMGB1) protein belongs to late inflammatory phase cytokine which shows a crucial role in the pathology of chronic pulmonary inflammatory diseases. Epithelial–mesenchymal transition (EMT) is implicated in airway remodeling and lung dysfunction in chronic inflammatory airway diseases. At present, the effects and mechanisms of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we found that HMGB1 treatment modulated EMT-related gene expression in human primary airway epithelial cells by RNA sequencing assay. HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. To dissect the mechanisms of HMGB1-induced EMT in human airway epithelial cells, we used western blotting, RNA interference and chemical inhibitors to investigate. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. HMGB1 caused AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Additionally, HMGB1 upregulated expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA attenuated HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that extracellular HMGB1 induced EMT through RAGE and β-catenin signaling pathway which might deteriorate bronchial epithelium which enhance disease severity and steroid resistance of airway.

Curcumin and epithelial-mesenchymal transition in breast cancer cells transformed by low doses of radiation and estrogen.

Breast cancer is a major cause of global mortality in women. Curcumin exerts anti-proliferative, anti-migratory and apoptotic effects. The aim of this study was to evaluate gene expression involved in epithelial-mesenchymal transition (EMT). An invitro model was developed with the MCF-10F immortalized breast epithelial cell line exposed to low radiation doses of high LET (linear energy transfer) α-particles (150keV/µm) and cultured in the presence of 17β-estradiol (estrogen). The following cell lines were used: i) MCF-10F, normal; ii) Alpha5, pre-tumorigenic, and iii) Tumor2 derived from Alpha5 injected into the nude mice. Our previous results have shown that Alpha5 and Tumor2 increased cell proliferation, anchorage independency, invasive capabilities and tumor formation in nude mice in comparison to control. Results indicated that curcumin decreased expression of EMT-related genes in Tumor2 cell line when compared to its counterpart as E-cadherin, N-cadherin, ZEB2, Twist1, Slug, Axl, vimentin, STAT-3, fibronectin; and genes p53 and caveolin-1, as well as apoptotic genes caspase-3, caspase-8, and others such as cyclinD1 and NFκB. All these changes induced a decrease in migratory and invasive capabilities of such a cell line. Thus, it seems that curcumin may impinge upon apoptosis and metastatic properties of the malignant cells exerting antitumor activity in breast cancer cells transformed by low doses of α-particles and estrogen invitro.

Downregulation of microRNA-122 promotes proliferation, migration, and invasion of human hepatocellular carcinoma cells by activating epithelial-mesenchymal transition.

ObjectiveTo investigate the effects of microRNA-122 (miR-122) on proliferation, migration, and invasion in human hepatocellular carcinoma (HCC) cells by activating epithelial–mesenchymal transition (EMT) pathways.MethodsmiR-122 mimics, miR-122 inhibitors, relevant control oligonucleotides, and Wnt1 were transfected into HepG2 and huh7 cell lines which were then divided into six groups: miR-122 group, anti-miR-122 group, miR-negative control (NC) group, anti-miR-NC group, miR-122 + Wnt1 group, and miR-122 + vector group. The miR-122 expressions and mRNA expressions of Wnt1 and EMT-related genes (E-cadherin, vimentin, β-cadherin, and N-cadherin) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression levels of Wnt1, E-cadherin, vimentin, β-cadherin, and N-cadherin were measured by Western blot. Cell proliferation, migration, and invasion were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound-healing assay, and Transwell assay, respectively.ResultsDual luciferase reporter gene results showed that Wnt1 is a direct target gene of miR-122 in both HepG2 and huh7 cell lines. Compared to miR-NC, anti-miR-NC, and miR-122 + Wnt1 groups, miR-122 expression was markedly higher in the miR-122 group and miR-122 + vector group, but was sharply decreased in anti-miR-122 group (both P<0.05), and the mRNA and protein levels of Wnt1, vimentin, β-cadherin, and N-cadherin decreased significantly; also E-cadherin increased, and cell proliferation, migration, and invasion decreased in the miR-122 group and miR-122 + vector group (all P<0.05), but the situation was totally reversed in the anti-miR-122 group (all P<0.05).ConclusionDownregulation of miR-122 promoted proliferation, migration, and invasion of human HCC cells by targeting Wnt1 and regulating Wnt/β-catenin pathway which activated the EMT pathways.

Prostate-derived ets factor represses tumorigenesis and modulates epithelial-to-mesenchymal transition in bladder carcinoma cells

Prostate-derived Ets (E-twenty six) factor (PDEF), an epithelium-specific member of the Ets family of transcription factors, has been shown to play a role in suppressing the development of many epithelium-derived cancers such as prostate and breast cancer. It is not clear, however, whether PDEF is involved in the development or progression of bladder cancer. In a comparison between normal urothelium and bladder tumor tissue, we identified significant decreases of PDEF in the tumor tissue. Further, the immunohistochemistry assays indicated a significantly higher immunostaining of PDEF in low-grade bladder tumors. Additionally, the highly differentiated transitional-cell bladder carcinoma RT-4 cells expressed significantly more PDEF levels than the bladder carcinoma HT1376 and the T24 cells. Ectopic overexpression of PDEF attenuated proliferation, invasion, and tumorigenesis of bladder carcinoma cells in vitro and in vivo. PDEF enhanced the expression levels of mammary serine protease inhibitor (MASPIN), N-myc downstream regulated gene 1 (NDRG1), KAI1, and B-cell translocation gene 2 (BTG2). PDEF modulated epithelial-mesenchymal-transition (EMT) by upregulating E-cadherin expression and downregulating the expression of N-cadherin, SNAIL, SLUG, and vimentin, leading to lower migration and invasion abilities of bladder carcinoma cells. Filamentous actin (F-actin) polarization and remodeling were observed in PDEF-knockdown RT-4 cells. Our results suggest that PDEF gene expression is associated with the extent of bladder neoplasia and PDEF modulated the expressions of EMT-related genes. The induction of BTG2, NDRG1, MASPIN, and KAI1 gene expressions by PDEF may explain the inhibitory functions of PDEF on the proliferation, invasion, and tumorigenesis in bladder carcinoma cells.

The long non-coding RNA TUG1 indicates a poor prognosis for colorectal cancer and promotes metastasis by affecting epithelial-mesenchymal transition.

BackgroundLong intergenic non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are involved in gene expression regulation. Taurine up-regulated gene 1 (TUG1) is a cancer progression related lncRNA in some tumor oncogenesis; however, its role in colorectal cancer (CRC) remains unclear. In this study, we determined the expression patterns of TUG1 in CRC patients and explored its effect on CRC cell metastasis using cultured representative CRC cell lines.MethodsThe expression levels of TUG1 in 120 CRC patients and CRC cells were determined using quantitative real-time PCR. HDACs and epithelial-mesenchymal transition (EMT)-related gene expression were determined using western blot. CRC cell metastasis was assessed by colony formation, migration assay and invasion assay.ResultsOur data showed that the levels of TUG1 were upregulated in both CRC cell lines and primary CRC clinical samples. TUG1 upregulation was closely correlated with the survival time of CRC patients. Overexpression of TUG1 in CRC cells increased their colony formation, migration, and invasion invitro and promoted their metastatic potential in vivo, whereas knockdown of TUG1 inhibited the colony formation, migration, and invasion of CRC cells invitro. It is also worth pointing out that TUG1 activated EMT-related gene expression.ConclusionOur data suggest that tumor expression of lncRNA TUG1 plays a critical role in CRC metastasis. TUG1 may have potential roles as a biomarker and/or a therapeutic target in colorectal cancer.

High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

Epithelial–mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway.

Krüppel-like factor 17, a novel tumor suppressor: its low expression is involved in cancer metastasis.

Krüppel-like factor (KLF) family is highly conserved zinc finger transcription factors that regulate cell proliferation, differentiation, apoptosis, and migration. KLF17 is a member of the KLF family. Recent studies have demonstrated that KLF17 low expression and inactivation are caused by microRNA, gene mutation, and loss of heterozygosity in human tumors, which participates in tumor progression. KLF17 low expression increases cancer metastatic viability; its mechanism is that low KLF17 mediates epithelial-mesenchymal transition (EMT) through regulating EMT-related genes expression; the reduced-KLF17 also increases cancer metastasis though upregulating inhibitor of DNA binding 1 (ID1). Additionally, mutant p53 proteins are capable of developing a complex with KLF17, which mediate the depletion of KLF17 inhibiting EMT gene transcription and increases cancer metastasis. KLF17 downregulation also mediates the activation of TGF-β pathway.

Activation of pancreatic stellate cells involves an EMT-like process.

Pancreatic adenocarcinoma (PDAC) and chronic pancreatitis (CP) are characterized by a desmoplastic reaction involving activated pancreatic stellate cells (PSCs). However, the mechanisms of PSC activation remain poorly understood. We examined whether the epithelial-mesenchymal transition (EMT) process might play a role in PSC activation. PSCs were isolated from a rat pancreas and characterized using immunofluorescence and immunocytochemistry. We evaluated changes in cell motility and in the expression levels of a panel of EMT-related genes during the PSC activation process. Activation of PSCs occurred after 48 h of in vitro culture, as indicated by a morphological change to a myofibroblastic shape and a decrease in the number of cytoplasmic lipid droplets. After activation, PSCs showed enhanced cell migration ability compared to quiescent cells. In addition, the expression of epithelial markers (E-cadherin, BMP7 and desmoplakin) decreased, while expression of mesenchymal markers (N-cadherin, vimentin, fibronectin1, collagen1α1 and S100A4) increased in activated PSCs. EMT-related transcription factors (Snail and Slug) were also upregulated after PSC activation. The concurrent increase in cell migration ability and alterations in EMT-related gene expression suggests that the activation of PSCs involves an EMT-like process. The knowledge that PSC activation involves an EMT‑like process may help to identify potential new therapeutic targets to alleviate pancreatic fibrosis in diseases like CP and PDAC.

64 EPITHELIAL MESENCHYMAL TRANSITION AND DIFFERENTIATION OF STEROIDOGENIC FACTOR 1 MOUSE EMBRYONIC STEM CELLS INTO THE STEROIDOGENIC CELLS

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we transfected mouse embryonic stem cells (mESCs) with the mouse SF1 gene (SF1-mESCs) by using the nucleofector (Lonza), and selected SF1-mESCs by G418 250 μg mL–1. The selected cells were differentiated into granulosa-like cells through hanging-drops for 3 days, suspension culture for 1 day, then attachment onto 6-well plates. Expression of steroidogenesis-related genes and gonadal lineage-markers was analysed by real-time PCR. To test the phenotype for granulosa-like cells, transcripts of specific forkhead transcription factor (Foxl2) and follicle stimulating hormone receptor (Fshr) were measured. Also, expression of EMT-related genes, such as E-Cadherin (Cdh1), N-Cadherin (Cdh2), Snai1, Snai2 (Slug), Twist, and Vimentin, was monitored. SF1-mESCs were differentiated into the primitive streak‐mesendoderm and the steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (Hsd3b1), cytochrome P450-containing enzyme (Cyp)-11a1, and Cyp19a1 were time-dependently changed. Next, the mRNA levels of Foxl2 and Fshr representing granulosa-like cell were increased during differentiation of SF1-mESCs. Especially, the level of oestradiol and Cdh2 was increased at a specific differentiation time. We induced differentiation of mESCs into the functional granulosa-like cells through transfection of the mouse SF1 gene. These cells will be useful for further study and potential application of these cells in steroidogenesis. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

Allo immune response, epithelial mesenchymal transition and chronic allograft fibrosis – Is there a link?

Introduction Mechanisms of chronic rejection manifested by irreversible fibrosis remain unclear. Phenotypic conversion of renal tubular epithelial and endothelial cells of the allograft to mesenchymal cells [Epithelial/Endothelial to Mesenchymal Transitioning (EMT/EndOEMT)] in the onset of fibrosis in the transplanted organ is proposed hypothesis. Objectives To detect EMT by way of gene expression profiling related to EMT using qualitative CR and quantitative real-time PCR covering 84 genes related to EMT. Materials: Anti-HLA Class I chimeric monoclonal antibody to the constant region of HLA Class I antigen (Invivogen, San Diego CA), EMR8-5: (Abcam ® ,Cambrige MA), a monoclonal HLA-Class I antibody that reacts with the heavy chains of Human HLA Class I A, B, and C and Pooled Positive Serum (PPS) containing antibodies to all Class I and Class II HLA allo-antigen and some possible non-HLA antigens. Renal Epithelial Cell (REC) and Human Umbilical Endothelial Cell (HUEC) lines (Life Line Cell Technologies, Frederick MD) were used. A total of 37 paraffin embedded renal allograft biopsy samples with varying etiologies and varying percentages of fibrosis were retrieved from Rush University Medical Center in Chicago, IL. An EMT RT 2 profiler PCR Array (Qiagen) specific for 84 EMT-related genes was used to assess the expression of these genes in 12 renal allograft biopsies and 2 cell cultures Results and conclusions The EMT Gene array profiling by realtime PCR indicated that in both in vitro cell cultures and biopsies there was significant changes in EMT related gene expressions. The overall gene expression pattern revealed up regulation of several EMT related genes in renal epithelial cells, HUEC, and in biopsy samples. The biopsy sample findings on EMT related gene expression array point in the direction of contributing towards finding a molecular signature of chronic allograft rejection.

Biodegradable interstitial release polymer loading a novel small molecule targeting Axl receptor tyrosine kinase and reducing brain tumour migration and invasion.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. The neoplasms are difficult to resect entirely because of their highly infiltration property and leading to the tumour edge is unclear. Gliadel wafer has been used as an intracerebral drug delivery system to eliminate the residual tumour. However, because of its local low concentration and short diffusion distance, patient survival improves non-significantly. Axl is an essential regulator in cancer metastasis and patient survival. In this study, we developed a controlled-release polyanhydride polymer loading a novel small molecule, n-butylidenephthalide (BP), which is not only increasing local drug concentration and extending its diffusion distance but also reducing tumour invasion, mediated by reducing Axl expression. First, we determined that BP inhibited the expression of Axl in a dose- and time-dependent manner and reduced the migratory and invasive capabilities of GBM cells. In addition, BP downregulated matrix metalloproteinase activity, which is involved in cancer cell invasion. Furthermore, we demonstrated that BP regulated Axl via the extracellular signal-regulated kinases pathway. Epithelial-to-mesenchymal transition (EMT) is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression; we demonstrated that BP reduced the expression of EMT-related genes. Furthermore, we used the overexpression of Axl in GBM cells to prove that Axl is a crucial target in the inhibition of GBM EMT, migration and invasion. In an in vivo study, we demonstrated that BP inhibited tumour growth and suppressed Axl expression in a dose-dependent manner according to a subcutaneous tumour model. Most importantly, in an intracranial tumour model with BP wafer in situ treatment, we demonstrated that the BP wafer not only significantly increased the survival rate but also decreased Axl expression, and inhibited tumour invasion. These results contribute to the development of a BP wafer for a novel therapeutic strategy for treating GBM invasion and increasing survival in clinical subjects.

Abstract 5189: Inhibitory effect of phytochemicals on oncostatin-M-induced tumor invasion and migration in human esophageal carcinoma

Abstract An epithelial-to-mesenchymal transition (EMT) is one of important phenomenon in tumor metastasis. Oncostatin-M (OSM) belonging to the IL-6 family is a cytokine that participates in invasion and migration of various cancer cells. It is produced by macrophage and stimulates the JAK/STAT signal transduction pathway. In this study, we examined the effect of OSM on EMT of human esophageal carcinoma cells and found a way to inhibit cell metastasis. We first observed morphological changes of tumor cells from an epithelial-like phenotype to a more spindle-shaped, mesenchymal phenotype by OSM. Consistent with these findings, OSM also promoted cell motility using a wound scratch assay and matrigel invasion assay. Moreover, we observed that intracellular stat3 phosphorylation was promoted by the stimulation of OSM by Western blotting. To confirm the contribution of p-stat3, we used a selective JAK2/STAT3 inhibitor cucurbitacin I. As a result, cucurbitacin I strongly inhibited OSM-induced tumor cell invasion and migration. Then, we used two dietary phytochemical resveratrol (polyphenol in grapes) and sulforaphane (isothiocyanate in broccolis), both were reported as candidates of stat3 inhibitor. By the addition of resveratrol or sulforaphane, we found that these markedly inhibited OSM-induced stat3 phosphorylation, the expression of EMT-related genes (snail, slug and vimentin) and tumor cell migration. Overall, our findings provide new evidence that these phytochemicals suppress OSM-induced tumor invasion and migration through inhibiting stat3 phosphorylation and EMT pathway. Citation Format: Eijin Naka, Yuko Takahashi, Hisahiro Matsubara, Kazunori Kato. Inhibitory effect of phytochemicals on oncostatin-M-induced tumor invasion and migration in human esophageal carcinoma. [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 5189. doi:10.1158/1538-7445.AM2015-5189

Abstract 2981: The epithelial to mesenchymal transition (EMT) produces colorectal cancer subpopulations with strikingly different mutation profiles

Abstract The epithelial-mesenchymal transition (EMT) has been well-recognized as an important mechanism promoting cancer cell invasion and “stemness”, metastasis and therapeutic resistance. While the EMT-related differential gene expression has been extensively elucidated, genetic understanding of this process in cancer is largely lacking. Using unsupervised analysis, we previously identified an “intrinsic” differential gene expression signature in colorectal cancer (CRC) that was prognostic and highly correlated (p = 10−134) to EMT. Thus, we hypothesized that whole exome sequencing (WES), guided by gene expression phenotype, might identify mutations underpinning the EMT program. We first sorted 2144 CRC tumors based on their assigned degree (scores) of EMT. Ten tumors from the bottom 15% of tumors and nine tumors from the top 15% of tumors were selected, with paired normal control tissues. The WES data were then processed to identify non-synonymous SNVs, indels and LOH. We observed a striking difference in the mean number of variation per tumor (p&amp;lt;10−4) for those with low (epithelial-like) vs. high (mesenchymal-like) EMT scores (low = 282 vs. high = 3). Further analysis suggests that there may be two subpopulations in the low EMT tumors − those with &amp;gt;400 mutations (4/10, avg. 608) that were subsequently confirmed for microsatellite instability (MSI) status through BAT testing where indels in TGFBR2 were seen often with BRAF mutations (3/4); those with 12-144 mutations (6/10, avg. 65), which still demonstrated significant increases in mutation accounts compared to the high EMT tumors (0-5 mutations, avg. 2.8). Surprisingly, unlike their low EMT counterparts, the high EMT tumors displayed almost no known driver gene mutations (except a TP53 mutation observed in one high EMT sample with p&amp;lt;0.01). This potentially important finding was confirmed by analysis of another 468 CRC tumors that underwent targeted exome sequencing of 1321 cancer-related genes; that is, tumors in the subgroup lacking mutations of common driver genes such as APC (truncated mutations), KRAS, TP53 and BRAF (V600E) had significantly higher EMT scores compared to other subgroups. Moreover, WES identified a limited number of mutated genes in high EMT tumors, among which FAM186A was the most frequently mutated gene with p&amp;lt;10−4. Alternately, RNAseq was also performed on five high and three low EMT samples for whole transcriptome analysis, which identified a list of the most deregulated genes, isoforms and coding sequences (e.g. SPP1, THBS2, THBS4 and SNAIL1 upregulated in high EMT tumors) as well as novel mutations (e.g. CDCP1 that was reported as a cell surface marker of migrating colon cancer stem cells). Therefore, our next-generation sequencing analysis has discovered the most mesenchymal-like CRCs as a novel, genetically distinct subpopulation lacking common driver mutations. Citation Format: Mingli Yang, Michael J. Schell, Norman H. Lee, Timothy J. Yeatman. The epithelial to mesenchymal transition (EMT) produces colorectal cancer subpopulations with strikingly different mutation profiles. [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 2981. doi:10.1158/1538-7445.AM2015-2981

Abstract 2370: Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition

Abstract During tumor formation, normal tissue microenvironment is transformed in an “altered” niche, composed of non-malignant supporting cells, which influence the homeostasis of cancer cells via paracrine regulators. Tumor-associated stromal cells (TASC) are the prominent stromal elements in most types of human carcinomas including colorectal cancer (CRC). The differentiation of TASC from other cell types, such as resident stromal cells or bone marrow-derived mesenchymal stem cells (BM-MSC) is mainly mediated by factors produced during the crosstalk with malignant cells. TASC produce various extracellular matrix proteins, chemokines, and other tumor-promoting factors which affect vascularization, proliferation, tumor cell invasiveness and survival. Emerging data suggests that they also play a critical role in determining response to therapy. For instance TASC-derived factors may contribute to the development of a protective milieu by influencing cell-cell/cell-matrix interactions, cancer cell survival, and suppression of anti-tumor immune responses. Moreover, physical contact between TASC and malignant cells supports tumor cell survival via activation of anti-apoptotic pathways or inducing epithelial-to-mesenchymal transition (EMT). We previously showed that BM-MSC are capable of triggering EMT in CRC cells, leading to increased tumor cell aggressiveness in vitro and in vivo. However, the role played by the stromal component and the processes induced on CRC cells remain to be fully elucidated. For this purpose we have addressed phenotypic and functional characterization of TASC in vitro and we have analyzed TASC-mediated effects on CRC development and progression in vivo. TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity was assessed upon injection in NOD/SCID mice and developing tumors were analyzed. Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells they express membrane-bound TGF-β, through which they are capable to initiate EMT in tumor cells. Moreover CRC cells acquire a more invasive phenotype after co-culture with TASC. Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured with TASC show a significantly faster growth kinetic and develop significantly larger tumor masses as compared to tumor cells alone. Thus our data show that the stromal component of CRC increases the tumor cells malignancy triggering EMT induction. Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Raoul Droeser, Daniel Oertli, Markus Zuber, Raffaele Rosso, Ivan Martin, Michael Heberer, Giulio C. Spagnoli, Giandomenica Iezzi. Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition. [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 2370. doi:10.1158/1538-7445.AM2015-2370

Abstract 4073: Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties

Abstract Androgen receptor- (AR-) expressing prostate cancer (PCa) cell lines can transdifferentiate to neuro-ectodermal cell lineages under androgen deprivation. We have proposed that this transdifferentiation is mediated by initial regression to an unstable cancer stem cell-like (CSCL) intermediate state and we have developed a serum- and androgen-free custom cell culture medium that now allows us to efficiently reprogram PCa cells to this CSCL state and expand them for characterization. Exposure of adherent cultures of parental LNCaP, VCaP, LAPC4 or CWR22rv1 cells to the reprogramming medium induced a rapid and efficient (&amp;gt;67%) morphological change of plated cells to smaller, rounded cells with large nuclei and prominent nucleoli that grow in 3-dimensions. Following transfer of the reprogrammed cells they, thereafter, grow as cell rosettes that will form larger spheroids. Analysis of changes in gene expression by microarray gene expression profiling or immunohistochemistry (IHC) showed a remarkable overexpression of stem cell genes in the reprogrammed cells and a change in cell surface CD protein expression that resembles neural stem cells. Reprogramming drastically reduced expression of AR RNA and protein in all cell lines. Once reprogrammed, the CSCLs have significantly higher tumor-initiating capacity based upon tumors formed from cell serial dilutions xenografted into intact or castrated male nude mice. In vitro, reprogrammed cells were significantly more resistant to the anti-androgen enzalutamide and to hypoxia compared to parentals. By placing CSCLs into specific differentiation mediums, we could differentiate them back into neural-, oligodendrocyte-, glia- or osteoblast-like cells based upon induced expression of lineage-specific RNAs/proteins or return them to prostate-like cells (that re-express AR) by supplementing them with serum and androgen. Reprogrammed cells have an EMT phenotype based on increased expression of EMT-related genes and significantly increased invasiveness in in vitro and in vivo models. Comparison of gene expression changes acquired by reprogrammed CSCLs amongst the different cell models revealed a shared set of 65 over-/under-expressed genes (≥1.8-fold) between them. Finally, we have found that growth of parental LNCaP cells in androgen-deprived or enzalutamide-supplemented (serum-containing) growth medium increased the population of cells bearing CSCL surface CD stem markers. In summary, our studies show that commonly used PCa cell lines have the ability to efficiently transition between a differentiated and a CSCL state in an appropriate microenvironment that is depleted of androgens. While in the CSCL state, these cells show increased invasiveness, increased resistance to hormonal therapeutics and a propensity to transdifferentiate to alternate cell lineages under appropriate stimulation. Citation Format: Josselin Caradec, Amy A. Lubik, Mannan Nouri, Na Li, Manuel Altimarano-Dimas, Jennifer Bishop, Mani Moniri, Down Cochrane, Martin Gleave, Ralph Buttyan. Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties. [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 4073. doi:10.1158/1538-7445.AM2015-4073

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

MicroRNA-129-5p modulates epithelial-to-mesenchymal transition by targeting SIP1 and SOX4 during peritoneal dialysis

Peritoneal dialysis (PD) is the most readily feasible home-dialysis method for renal replacement therapy. However, repeated use of PD can lead to induction of mesothelial/epithelial–mesenchymal transition (MMT/EMT) and fibrosis, eventually leading to ultrafiltration failure and discontinuation of PD. MicroRNA-129-5p (miR-129-5p) is believed to be a potent downstream inhibitor of TGF-β1 in renal fibrosis, but the effect of miR-129-5p on MMT/EMT relevant to PD is unknown. In this study, as determined by microRNA array analysis and confirmed by northern blot analysis and real-time PCR, we demonstrate that miRNA-129-5p is decreased in mesothelial cells isolated from effluent of patients having PD for more than 6 months extending to several years compared with those who have undergone PD for less than 6 months. The decreased expression of miR-129-5p was accompanied with alterations in EMT-related genes and the expression of respective proteins in vivo. In addition, in in vitro studies we noted that the expression of E-cadherin and claudin-1 were significantly reduced with increased cell migration in HMrSV5, a human peritoneal mesothelial cell line (HPMC), treated with TGF-β1, whereas expression of vimentin, fibronectin and transcription factors SIP1 and SOX4 increased significantly, as assessed by real-time PCR, western blot analysis and immunofluorescence microscopy. Furthermore, alteration in EMT-related genes and proteins were reversed by overexpression of miR-129-5p. No effect was observed in cells treated with miR-negative control. Meanwhile, inhibition of SIP1 and SOX4 with their respective siRNA also could decrease the expression of EMT-related genes and protein levels in HPMCs induced with TGF-β1. Finally, we demonstrate that SIP1 can inhibit the promoter activity of E-cadherin while enhancing the promoter activity of vimentin. We also observed that miR-129-5p could directly target the 3′UTR of SIP1 and SOX4 genes, and repressed their post-transcriptional activities. These data suggest that there is a novel TGF-β1/miR-129-5p/SIP-1 or SOX4 pathway that has a significant role in MMT and fibrosis in the setting of PD.