Epithelial-mesenchimal Transition Research Articles

Investigation of the Effects of Juglone-Selenium Treatments on Epithelial-Mesenchimal Transition and Migration in BxPC-3 and PANC-1 Human Pancreatic Cancer Cells

Investigation of the Effects of Juglone-Selenium Treatments on Epithelial-Mesenchimal Transition and Migration in BxPC-3 and PANC-1 Human Pancreatic Cancer Cells

Peculiarities of epithelial-mesenchimal transition in endometrial carcinomas.

Epithelial-mesenchymal transition is an important component of tumor progression, due to which the cells of malignant neoplasms acquire invasive and migratory properties. Analysis of the literature and our own data show that the activation of proteins involved in epithelial-mesenchymal transition crucially affects the progression of endometrioid carcinoma of the endometrium and the significant variability of their expression could determine the clinical and morphological heterogeneity of this cancer. The most aggressive endometrioid carcinomas of the endometrium are characterized by a hybrid epithelial-mesenchymal phenotype, which is often associated with a collective type of invasion of endometrial tumor cells into the myometrium.

Circulation of tumor cells to destroy correlations in immune system of colorectal cancer patients.

e15508 Background: The most important stages of metastatic cascade are extravasation and invasion of malignant cells and their surviving in blood. The vast majority of circulating tumor cells (CTC) is destroyed by immune cells. The role of immune system which is able to play not only antitumor but also prooncogenic role is manifested both on local and systemic levels. We studied correlations between lymphocyte subsets` content in blood and in tumor of colorectal cancer patients (II, III, IV stages) with and without CTC. Methods: 60 colorectal cancer patients with II (n = 20), III (n = 20) and IV (n = 20) stages underwent surgery without previous chemotherapy. CTC were measured in blood by CellSearchSystem™ (JanssenDiagnostics, LLC), cell-mediated immunity was assessed in blood by flow cytometry (BD Canto II) and in tissue after surgery by immunohistochemistry; some markers of proliferation and epithelial-mesenchimal transition (EMT) in tumor cells (Ki-67, E- and N-cadherins) were also studied. Criteria of CTC-positive and CTC-negative samples were > 3 and ≤3 respectively. Correlative analysis was performed between the data of the patients with and without CTC in each stage, r was counted. Results: In CTC-positive patients with all the stages the number of strong and moderate correlations between system immunologic factors involving CD8+ appeared to be fewer than in CTC-negative ones (7 vs 19). On the contrary, the number of correlations with T regs in CTC-positive was increased: 5 vs 3 in CTC-negative patients. In patients with CTC > 3 fewer correlations were noted between factors of local and systemic immunity than in CTC-negative ones (9 vs 4 in II stage) and total disruption of all the correlations between system immunologic factors and proliferating tumor cells in III and IV stages was established. Some pathologic correlations appeared in CTC-positive patients like moderate direct one between activated T-lymphocytes` amount and Ki-67+ tumor cells. The number of correlations between intratumoral lymphocytes and tumor cells expressing proliferation and EMT markers in CTC-positive patients was decreased in comparison with CTC-negative ones (4 vs 10 in II stage, 1 vs 9 in III, 2 vs 9 in IV stage). Conclusions: The presence of CTC in colorectal cancer patients rather than tumor stage is associated with imbalance of their systemic and local immunologic factors. This provides some evidence that disruption of interactions in the immune system is at least partly due to CTC.

Lichen planus of anogenital region: clinical spectrum and approaches to therapy

Lichen planus is among the most common chronic anogenital noninfectious dermatoses both in male and female. The disease is characterized by clinical polymorphism, frequent involvement of skin and oral mucosa and protracted course. Typical, hypertrophic and erosive forms are distinguished. Erosive variant is commonly encountered in women and has a prominent tendency for scarring. The most common anogenital form in men is the typical lichen planus, which usually resolves completely. Vulvovagino-gingival and penogingival lichen planus are rare severe disease forms. Evolutional and biological heterogeny of clinical manifestations complicates the disease diagnosis. Lichen planus pathogenesis remains unclear. Pronounced scarring tendency in anogenital cases is believed to be the consequence of epithelial-mesenchimal transition. High-quality clinical trials of various therapeutic modalities in anogenital lichen planus are lacking. Management of such patients is mostly based on case series reports, practical experience and general principles of dermatologic treatment. This review focuses on contemporary views on clinical presentation, pathogenesis, diagnosis and approach to therapy of anogenital lichen planus.

Abstract 1794: Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted therapy

Abstract Introduction: Targeted therapies have recently revolutionized the treatment of BRAFV600-mutant metastatic cutaneous melanoma (CM). However, most patients do not achieve tumor regression at all due to the occurrence of resistance mechanisms. Mitogen-activated protein kinase (MAPK) pathway activation is a central step in BRAFV600-mutant CM pathogenesis and a better understanding of molecular mechanisms involved MAPK modulation is necessary to develop more effective therapeutic strategies. In the last years, Spry1 has been frequently described as an upstream regulator of MAPK signaling pathway, but its specific role in BRAFV600-mutant CM is still poorly defined. Materials and methods: Autologous BRAFV600-mutant CM cell cultures were generated from CM patients resected in our institution. The expression of Spry1 was evaluated by quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses. Spry1 gene was knock out using CRISPR. RNA-seq identified mRNAs commonly differentially expressed in Spry1 knock-out (Spry1KO) clones, and their expression was further validated by qRT-PCR. WB analyses evaluated the modulation of MAPK signaling pathways in Spry1KO clones. Cell proliferation was measured using xCELLigence instrument, whereas cell cycle progression, ROS induction, and apoptosis were assessed by flow cytometry. In vivo effects of Spry1 silencing were investigated in xenograft mice treated with or without BRAF inhibitor (BRAFi). Results: The expression of Spry1 in human CM was explored using publicly available cancer gene expression profiling and transcriptome sequencing data. mRNA expression of Spry1 was significantly elevated in metastatic CM respect to primary tumors and mRNA levels of Spry1 were significantly up-regulated in metastatic CM compared with primary lesions. The function of Spry1 was evaluated by comparing three BRAFV600-mutant CM cell lines and the respective Spry1KO cells. We found that Spry1KO induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. Moreover Spry1KO was found to impair the expression of several markers of epithelial-mesenchimal transition. In addition, ERK1/2 was found hyperactivated together with p38 leading to an increase in basal ROS levels in Spry1KO clones. This suggests that BRAFV600-mutant CM may restrain the activity of Spry1 to avoid oncogenic stress and to enable tumor growth. Indeed, treatment with the BRAFi vemurafenib down-regulated Spry1 levels in parental CM cell lines, indicating that Spry1 expression is sustained by MAPK/ERK signaling pathway in a positive feedback loop that safeguards cells from the potentially toxic effects of ERK1/2 hyperactivation. Disruption of this feedback loop rendered Spry1KO cells more susceptible to apoptosis and markedly improved response to BRAFi both in vitro and in vivo, as a consequence of the detrimental effect of ERK1/2 hyperactivation observed upon Spry1 abrogation. Conclusions: All together, our data strongly suggest that targeting Spry1 might offer a treatment strategy for BRAFV600-mutant CM by inducing the toxic effects of ERK-mediated signaling. Citation Format: Barbara Montico, Francesca Colizzi, Giorgio Giurato, Aurora Rizzo, Annamaria Salvati, Lorena Baboci, Dania Benedetti, Eliana Pivetta, Alessia Covre, Michele Dal Bo, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1794.

Epithelial-mesenchimal transition and its relationship with leaky gut syndrome as possible step in pathogenesis of endometriosis

Epithelial-mesenchimal transition and its relationship with leaky gut syndrome as possible step in pathogenesis of endometriosis

Abstract 4137: Circulating TGFâ and TNFá in metastatic breast cancer patients treated with eribulin. The TRANSERI project

Abstract Background: Eribulin (E) is approved for the treatment of metastatic breast cancer (mBC) patients (pts) after failure of antracyclines and taxanes. E interferes with microtubule leading to apoptosis and G2/M cell cycle arrest. In human cancer cell lines and in mice, it reverses epithelial mesenchimal transition (EMT) and reduces metastases in mice. TGFβ, an immunosuppressive cytokine, acts as growth factor for cancer-associated fibroblasts (CAFs) and promotes EMT. TNFα synergizes with TGFβ to promote EMT. The TRANSERI study investigates the modifications of TGFβ and TNFα levels in 40 mBC pts treated with E. Methods: Plasma levels of TGFβ and TNFα were determined by ELISA assay at baseline, before cycle (C) 3, 5 and at disease progression in mBC pts treated with E at 1.23 mg/m2, d 1-8 every 21 d. Statistical analysis of the changes in the longitudinal samples was performed by GraphPad 5. Clinical outcome was monitored according to standard internal follow up procedure. Results: We report analyses in pts who completed 5 C or progressed before C 5. So far, we have evaluated TGFβ level in 34 pts and TNFα in 26 pts. The median (M) basal TGFβ value was higher in pts than in 9 healthy volunteers (201.9 pg/ml vs 115.1 respectively). At C 5, 14 pts had a decrease in TGFβ with a M value approaching the one of healthy controls (161.9 pg/ml vs 115.1 pg/ml respectively). 19 pts progressed before C 5. Overall 26/33 pts experienced progression. The M value of TGFβ in pts at progression was 293.4 pg/ml. A significant difference of TGFβ was observed between non progressed vs progressed pts (p=0.021). The M TNFα value at baseline was higher in pts than in healthy volunteers, even if in both groups it was close to the lower sensitivity cut-off of the assay. Altogether 20/26 pts experienced progression, that occurred before C 5 in 14 out of them.Intriguingly, at C 5, the TNFα / TGFβ ratio was significantly lower in pts who did not progressed compared to the value at progression (p=0.048). Conclusions: TGFβ levels changed during treatment with E and correlate with outcome. We are evaluating the role of tumor burden in modulating the TNFα / TGFβ ratio. Definitive data will be presented. Citation Format: Ornella Garrone, Cristiana Lo Nigro, Andrea Michelotti, Fillippo Montemurro, Anna Maria Vandone, Andrea Abbona, Matteo Paccagnella, Antonella Falletta, Elena Genua, Claudia De Angelis, Federica Tonissi, Paola Vanella. Circulating TGFâ and TNFá in metastatic breast cancer patients treated with eribulin. The TRANSERI project [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 4137.

Activity and molecular targets of pioglitazone via blockade of proliferation, invasiveness and bioenergetics in human NSCLC

BackgroundPioglitazone, a synthetic peroxisome proliferator activated receptor (PPAR-γ) ligand, is known as an antidiabetic drug included in the thiazolidinediones (TZDs) class. It regulates the lipid and glucose cell metabolism and recently a role in the inhibition of numerous cancer cell processes has been described.MethodsIn our work we investigate the anti-tumor effects of pioglitazone in in vitro models of non small cell lung cancer (NSCLC) and also, we generated ex-vivo three-dimensional (3D) cultures from human lung adenocarcinoma (ADK) as a model to test drug efficacy observed in vitro. The inhibitory effect of pioglitazone on cell proliferation, apoptosis and cell invasion in a panel of human NSCLC cell lines was evaluated by multiple assays.ResultsPioglitazone reduced proliferative and invasive abilities with an IC50 ranging between 5 and 10 μM and induced apoptosis of NSCLC cells. mRNA microarray expression profiling showed a down regulation of MAPK, Myc and Ras genes after treatment with pioglitazone; altered gene expression was confirmed by protein analysis in a dose-related reduction of survivin and phosphorylated proteins levels of MAPK pathway. Interestingly mRNA microarray analysis showed also that pioglitazone affects TGFβ pathway, which is important in the epithelial-to-mesenchimal transition (EMT) process, by down-regulating TGFβR1 and SMAD3 mRNA expression. In addition, extracellular acidification rate (ECAR) and a proportional reduction of markers of altered glucose metabolism in treated cells demonstrated also cell bioenergetics modulation by pioglitazone.ConclusionsData indicate that PPAR-γ agonists represent an attractive treatment tool and by suppression of cell growth (in vitro and ex vivo models) and of invasion via blockade of MAPK cascade and TGFβ/SMADs signaling, respectively, and its role in cancer bioenergetics and metabolism indicate that PPAR-γ agonists represent an attractive treatment tool for NSCLC.

Role of HIF-1 in Cancer Progression: Novel Insights. A Review.

Hypoxia is a condition always present in tumor environment owing to the fast growth of tumor cells not supported by adequate blood supply. There is increasing evidence that hypoxia plays an important role in cancer dormancy and cancer metabolism, increasing stemness activity and bringing about cancer initiation and progression. This condition may influence the production of hypoxia inducible factor (HIF) a helix transcription factor which is involved in carcinogenesis and tumor growth through the regulation of genes involved in angiogenesis, glycolytic metabolism and other biological mechanisms. In normoxia condition HIF is inactivated by prolyl hydroxylase enzymes (EGLN 1-3, also known as PHD 1-3) using oxygen as a substrate. Once hydroxilated it binds to a protein called Von Hippel Lindau protein (VHL) for its degradation, whereas in hypoxia condition stabilization and nuclear translocation occur, leading to oncogenes activation. It has got three isoforms HIF-1 HIF-2 and HIF-3. The most studied factor is HIF-1 which is a heterodimer consisting of two forms, the form α is expressed in manner oxygen dependent, the form β is expressed constitutively. Its presence in tumor microenvironment could foster among other the expression of VEGF, HGF, Met protoncogene which induces degradation of the extracellular matrix and TWIST gene, which is in turn involved in a mechanism of cancer cell metastasis called epithelial-mesenchimal transition(EMT). In this review, we summarize the most important findings in HIF action in different types of cancer focusing on its properties to induce tumor cell growth and highlighting its poor prognostic value in different cancers sites.

Hypoxia and serum deprivation induces glycan alterations in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is a major global public health problem. The lack of targeted therapy and the elevated mortality evidence the need for better knowledge of the tumor biology. Hypoxia and aberrant glycosylation are associated with advanced stages of malignancy, tumor progression and treatment resistance. Importantly, serum deprivation regulates the invasive phenotype and favors TNBC cell survival. However, in TNBC, the role of hypoxia and serum deprivation in the regulation of glycosylation remains largely unknown. The effects of hypoxia and serum deprivation on the expression of glycosyltransferases and glycan profile were evaluated in the MDA-MB-231 cell line. We showed that the overexpression of HIF-1α was accompanied by acquisition of epithelial-mesenchimal transition features. Significant upregulation of fucosyl- and sialyltransferases involved in the synthesis of tumor-associated carbohydrate antigens was observed together with changes in fucosylation and sialylation detected by Aleuria aurantia lectin and Sambucus nigra agglutinin lectin blots. Bioinformatic analysis further indicated a mechanism by which HIF-1α can regulate ST3GAL6 expression and the relationship within the intrinsic characteristics of TNBC tumors. In conclusion, our results showed the involvement of hypoxia and serum deprivation in glycosylation profile regulation of TNBC cells triggering breast cancer aggressive features and suggesting glycosylation as a potential diagnostic and therapeutic target.

The TRANSERI study: Effect of eribulin (E) on circulating TGFβ and TNFα in metastatic breast cancer (mBC) patients (pts)—Relationship with outcome.

e24129 Background: E is approved for the treatment of mBC pts after previous exposure to antracyclines and taxanes. E interferes with microtubule leading to apoptosis and cell cycle arrest in G2/M. E reverses epithelial mesenchimal transition (EMT) in human cancer cell lines and in mice. Moreover, reduces metastatization in mice. TGFβ is an immunosuppressive cytokine, is a growth factor for cancer-associated fibroblasts (CAFs) and promotes EMT. TNFα synergizes with TGFβ to promote EMT. EMT permits cancer cells trafficking thus driving metastatization. Our study investigates the interference among E and TGFβ and TNFα levels in pts and the correlation with the outcome and the metastatic spread. Methods: Blood levels of TGFβ and TNFα were determined at baseline, before cycle (C) 3, 5 and at disease progression in mBC pts treated with E at 1.23 mg/m2, d 1–8 every 21 d. Results: We report data on 24 pts who completed 5 C or progressed before C 5. We did not observe changes of TNFα during treatment. The median (M) basal TGFβ value was higher in pts than in 4 healthy volunteers (M concentrations: 232 pg/ml vs 114 pg/ml respectively). We divided our population according to basal TGFβ levels upper or lower the M value. M PFS was similar in the two groups (112 vs 100 d). TGFβ increased in 9 pts from basal to C 5, and decreased in 15 pts. We observed a numerical difference in M PFS between the pts with decreased and increased values (107 vs 82 d respectively). In 8 pts TGFβ decreased at each subsequent point. M PFS in these pts was 167 vs 84 d in the remaining. Notwithstanding the continuous decline of TGFβ, 2 of these pts progressed at C 5 (both at CNS). In pts with continuous TGFβ decline the M value approaches healthy controls (160 pg/ml [range 303-90] vs 114 pg/ml [range 120-85] respectively). At C 5 9 pts did not progress: 3 PR and 6 SD (M PFS 175 d). TGFβ decreased in all but 2 pts (1PR+1SD). In these 2 pts PD occurred as early as 21 and 30 d respectively from C 5. The behaviour of pts with SD suggests that there is no correlation between tumor burden and TGFβ level. Conclusions: Basal TGFβ does not predict outcome. TGFβ changed during treatment with E regardless of tumor load. Our results suggest that TGFβ changes correlate with outcome.

GATA factor genes in the Drosophila midgut embryo.

The Drosophila GATA factor gene serpent (srp) is required for the early differentiation of the anterior and posterior midgut primordia. In particular, srp is sufficient and necessary for the primordial gut cells to undertake an epithelial-to-mesenchimal transition (EMT). Two other GATA factor genes, dGATAe and grain (grn), are also specifically expressed in the midgut. On the one hand, dGATAe expression is activated by srp. Embryos homozygous for a deficiency uncovering dGATAe were shown to lack the expression of some differentiated midgut genes. Moreover, ectopic expression of dGATAe was sufficient to drive the expression of some of these differentiation marker genes, thus establishing the role of dGATAe in the regulation of their expression. However, due to the gross abnormalities associated with this deficiency, it was not possible to assess whether, similarly to srp, dGATAe might play a role in setting the midgut morphology. To further investigate this role we decided to generate a dGATAe mutant. On the other hand, grn is expressed in the midgut primordia around stage 11 and remains expressed until the end of embryogenesis. Yet, no midgut function has been described for grn. First, here we report that, as for dGATAe, midgut grn expression is dependent on srp; conversely, dGATAe and grn expression are independent of each other. Our results also indicate that, unlike srp, dGATAe and grn are not responsible for setting the general embryonic midgut morphology. We also show that the analysed midgut genes whose expression is lacking in embryos homozygous for a deficiency uncovering dGATAe are indeed dGATAe-dependent genes. Conversely, we do not find any midgut gene to be grn-dependent, with the exception of midgut repression of the proventriculus iroquois (iro) gene. In conclusion, our results clarify the expression patterns and function of the GATA factor genes expressed in the embryonic midgut.

Abstract P5-04-01: The TRANSERI project: Effect of eribulin (E) in patients with metastatic breast cancer (mBC) on circulating TGFβ and TNFα. Relationship with outcome

Abstract Background: E is approved for the treatment of mBC patients (pts) after failure of at least 2 previous chemotherapy (CT) regimens containing antracyclines and taxanes. Its mechanism of action interferes with microtubule leading to cell cycle arrest in G2/M phase and cell apoptosis. An in vitro study in triple negative BC cell lines shows that exposure to E reverses epithelial mesenchimal transition (EMT) phenotype toward an epithelial morphology and induces changes of gene profiling and protein expression. Accordingly in mice E reduces metastatization and can reverse EMT. TGFβ is an immunosuppressive cytokine and a growth factor for cancer-associated fibroblasts (CAFs). In addition it drives EMT. TNFα synergizes with TGFβ to promote EMT. While CAFs pave the way for metastatization, EMT permits cancer cells trafficking through the blood flow following CAFs and finally developing metastases. The purpose of the study is to investigate whether E interferes with TGFβ and TNFα levels and if the changes correlate with the outcome and the metastatic spread. Methods: Pts with mBC, after failure of at least 2 previous CT lines were treated with E delivered at 1.23 mg/m2, d 1–8 every 21 d. Blood levels of TGFβ and TNFα were determined at baseline, before cycle 3, 5 and at disease progression. The changes observed were correlated with the outcome and the metastatic spread. Results: The study is ongoing. Here we report preliminary data on 16 pts who completed 3 cycles of E. No change of TNFα level was observed during treatment. On the contrary, TGFβ levels changed during treatment. Basal levels of TGFβ were divided in upper or lower the median (m) value. We did not observe any difference in m PFS between high or low values (137 d vs 141). However the m TGFβ value in pts was much higher than that observed in 3 healthy volunteers (m concentrations: 205 pg/ml [C.I. 115-920] vs 108 pg/ml [C.I. 85-120] respectively). In 5 pts, TGFβ increased between cycle 1 and cycle 3, while diminished in 11 pts. We observed a numerical difference in PFS between the pts with decreased and increased values (150 d vs 85, p=NS). We then divided the population in 3 groups: pts with TGFβ increased more than 25% of their basal levels (increased), pts with changes between +25% and -25% compared to their basal levels (stable) and pts with decreased values more than 25% of their basal levels (decreased). Comparing “increased” vs “stable” + “decreased” we observed a trend toward longer PFS favouring the latter group (77 d vs 144 p=0.12). We collected the third determination in 14 pts. We did not analyse these data yet. However we observed that in 6 pts, TGFβ continues to decline. None of these pts progressed. In these pts the m value of TGFβ approaches healthy controls value (m concentrations: 180 pg/ml [C.I. 200-100] vs 108 pg/ml [C.I. 85-120]). Conclusions: TNFα does not change during E treatment. On the contrary, TGFβ changes compared to basal levels. In pts with increased TGFβ between cycle 1 and 3 the PFS is lower than that observed in pts with stable or decreased levels (p=0.12). Pts with continue decline of TGFβ at cycle 5, approach the values of the healthy volunteers. None of them progressed. Updated results will be presented. Citation Format: Garrone O, Tonissi F, Lingua D, Vandone AM, Vivenza D, Denaro N, Lo Nigro C, Merlano MC. The TRANSERI project: Effect of eribulin (E) in patients with metastatic breast cancer (mBC) on circulating TGFβ and TNFα. Relationship with outcome [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-04-01.

Recent Advances in Comprehending the Signaling Pathways Involved in the Progression of Breast Cancer.

This review describes recent advances in the comprehension of signaling pathways involved in breast cancer progression. Calcium sensing receptor (CaSR), caveolae signaling, signaling referred to hypoxia-inducing factors and disturbances in the apoptotic machinery are related to more general biological mechanisms and are considered first. The others refer to signaling pathways of more specific biological mechanisms, namely the heparin/heparin-sulfate interactome, over-expression of miRNA-378a-5p, restriction of luminal and basal epithelial cells, fatty-acid synthesis, molecular pathways related to epithelial to mesenchimal transition (EMT), HER-2/neu gene amplification and protein expression, and the expression of other members of the epithelial growth factor receptor family. This progress in basic research is fundamental to foster the ongoing efforts that use the new genotyping technologies, and aim at defining new prognostic and predictive biomarkers for a better personalized management of breast cancer disease.

Eribulin in Advanced Breast Cancer: Safety, Efficacy and New Perspectives

Eribulin is a synthetic analog of halichondrin B belonging to microtubule-targeted agents with a distinct mechanism of inhibition of microtubule dynamics. This molecule has multiple nonmitotic effects on tumor biology, exhibiting effects on epithelial-mesenchimal transition and tumor vasculature. We review here preclinical and clinical studies on eribulin. The mitotic and nonmitotic effects together with its favorable safety profile make eribulin a unique drug with high potential in the treatment of metastatic breast cancer. The new emphasis of eribulin mechanism of action on vascular remodeling, microenvironment modifications and reversal of epithelial-mesenchimal transition paves the way to rethinking the use of the drug in an immunological perspective.

Epithelial-to-Mesenchimal Transition (EMT) in experimental intestinal fibrosis

Inflammatory Bowel Diseases (IBD) are chronic and progressive inflammatory disorders that may result in intestinal fibrosis, characterized by excessive and uncon- trolled deposition of extracellular matrix components (ECM). Other than Smad- dependent the profibrotic effects of TGFβ signaling that leads to a phenotype switch of intestinal mesenchymal cells, resulting in proliferation and collagene deposition (1), source of fibrotic tissue comes from the process called Epithelial-to-Mesenchymal Transition (EMT), in which also epithelial cells acquire fibroblastic phenotype and related function (2). The purpose of our study was to evaluate the expression of pro- tein markers of EMT in a mouse model of intestinal fibrosis and their changes after the administration of the experimental drug GED- 0507-34 Levo, that is able to inhibit and revert the fibrotic phenotypic switch. Immunohistochemistry (IHC) and immunofluorescence (IF) evalutations for E-cadherin and β-catenin were performed in three groups of C57BL/6 mice: Dextran Sulphate Sodium (DSS) colitis group, DSS+GED group and controls. In the same groups E-cadherin, β-catenin, p-GSK3-beta and GSK3-beta were evaluated by immunoblotting. In IHC and IF analysis, was observed a significant decrease of E-cadherin and β-catenin expression in DSS group compared to control. GED administration was able to revert the DSS effect by increasing the expression of both proteins to levels similar to the control. Comparable results were obtained by immunoblotting. Furthermore the immunoblotting assay showed that the known EMT inhibitor GSK3-beta is activated in the DSS+GED group suggesting that GSK3-beta activity is required for the restoration of the epithelial phenotype exerted by GED. Taken together, our results indicates that the EMT is another cause of colla- gen deposition in our mouse model and thus it could be a valid target for the devel- opment of a treatment for intestinal fibrosis.

Roles of lncRNA in breast cancer

Epithelial-to-mesenchimal transition (EMT) is a process involved in invasion and metastasis of tumors. The occurrence of EMT during tumor progression resembles the developmental scenario and sheds light on important mechanisms for the initial step of metastasis - invasion where noninvasive tumor cells acquire motility and ultimately disseminate to distant organs. The hallmark of EMT is the loss of expression of the cell-cell adhesion molecule E-cadherin. The numerous reports by many authors as well as our own results indicate that E-cadherin plays a role in CNS tumors - meningiomas. Our studies showed that 73 % of meningiomas had downregulation of E-cadherin. Moreover, loss of heterozygosity of E-cadherin was observed in 32 % of meningiomas. Bound to E-cadherin in adherens junctions is beta-catenin, whose translocation to the nucleus is yet another molecular event involved in EMT. In our study beta-catenin was progressively upregulated from meningothelial to atypical, while 60 % of anaplastic meningiomas showed upregulation and nuclear localization of the protein. The elucidation of molecular mechanisms that govern EMT will offer new approaches and targets to restrain metastasis.

Abstract 3310: N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers

Abstract Introduction: Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance. Previously, different groups have suggested an association between chemoresistance and epithelial to mesenchimal transition (EMT) in certain advanced cancers. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with EMT, is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: Tissue microarrays of CRPC and high grade prostate cancer clinical samples were stained for TLR4. N-cadherin positive and negative cell lines were evaluated for sensitivity against docetaxel. N-cadherin and TLR4 were ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, VCaP). Docetaxel resistant cell lines (LNCaP-DocR and VCaP-DocR) were established through prolonged exposure to 10-20uM Docetaxel. For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. N-cadherin expressing cell lines were treated with 2A9 monoclonal antibody (mAb), which was raised against the extracellular domain of N-cadherin, and by N-cadherin siRNA knockdown. For in vivo studies, N-cadherin positive xenografts were treated with 2A9 mAb and docetaxel, and tumor growth monitored over time. Results: Tissue microarray results showed significant TLR4 expression in CRPC samples (p < 0.005). In vitro studies showed TLR4 induction correlated with N-cadherin expression. This upregulation of N-cadherin and TLR4 expression promoted docetaxel resistance in CRPC by the activation of NF-kB signaling. Monoclonal antibodies against N-cadherin and TLR4 potentiated docetaxel induced cytotoxicity. Docetaxel resistant cells exhibited EMT phenotypes with increased invasion and castration resistant growth. Treatment with 2A9 antibody restored sensitivity to docetaxel. Combination therapy with 2A9 monoclonal antibody and docetaxel significantly affected tumor growth in N-cadherin positive CRPC xenograft models. Conclusions: These studies suggest that N-cadherin promotes TLR4-dependent docetaxel resistance in CRPC. Although further investigation will be needed, therapeutic targeting of N-cadherin with monoclonal antibodies in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Citation Format: Tatsuya Shimomura, Evelyn Kono, Chau P. Tran, Joyce Yamashiro, Shu Lin, Sean Hyung-Kwon Lee, Zev A. Wainberg, Robert E. Reiter. N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers. [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 3310. doi:10.1158/1538-7445.AM2014-3310

A loss of microRNA expression as a characterization of synchronous peritoneal secondary localizations of epithelial ovarian cancer as compared to primary tumors.

11037 Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and one of the most challenging areas of cancer research being a highly heterogeneous disease difficult to diagnose and treat. EOC has a peculiar dissemination process due to the sloughing-off of cells from primary tumors and their spread throughout the peritoneal cavity. A better characterization of the mechanism involved in tumor spreading might help in design new therapeutic intervention. Methods: Forty-four couples of chemo naïf primary tumors and synchronous secondary peritoneal localizations, obtained at primary surgery from MITO2 clinical trial, have been profiled for microRNA (miRNA) expression on an Agilent Platform. Total RNA was extracted from formalin-fixed paraffin embedded tissues. An independent validation set of samples with similar characteristics, has been collected at INT Milan. Results: By class comparison analysis, imposing a false discovery rate <10%,45 miRNAs were identified as differentially expressed: 32 down-modulated and 13 up-modulated in secondary localizations compared to primary tumors. Among the miRNAs down-modulated in the secondary localizations we detected most of the miRNA belonging to the Xq27.3 cluster, whose low expression we previously described to be associated with EOC early relapse, and a number of miRNAs related to epithelial/mesenchimal transition (EMT) whose modulation could be related to dissemination of the disease and response to drug treatment. In particularly loss of has-miR-506 resulted associated to platinum resistance since its ectopic expression in EOC cell lines increased their sensitivity to the drug. Furthermore preliminary data indicated that has-miR-506 regulated N-cadherin linking its modulation to EMT. Conclusions: To our knowledge, the present study is the first attempt to characterize a miRNA signature differentially expressed between EOC primary tumors and synchronous secondary peritoneal localizations. The validation of the miRNA profile as well as of target genes might help in elucidating EOC dissemination mechanisms and in defining possible new therapeutic targets.

Sox2 induces Mesenchymal to Epithelial transition in somatic cells

During late embryonic development mesodermal cells generate epithelial organs, such as kidney and ovary, via a process named Mesenchymal to Epithelial Transition (MET) (1). This process can also occur reversely giving rise to Epithelial to Mesenchimal transition (EMT) (2), which represents the mechanism by which metastasis originate from primary tumors. Many lines of evidence underlie a continuous interaction between these two programs. Indeed MET is believed to participate in the establishment and stabilization of distant metastases by allowing cancerous cells to regain epithelial properties and integrate into distant organs. It has been shown that fibroblasts undergo MET at some point during reprogramming to mimic undifferentiated ESCs (2).Since SOX2 is an essential factor to reprogram somatic cells to stem cell fate, we investigated if it might play a role in inducing MET in mouse embryonic fibroblasts. Using a lentiviral vector we found that Sox2 transduced cells changed morphology as early as two days after infection compared to their mock infected controls. Although cells showed an epithelial-like morphology, they were E-cadherin negative. Furthermore, Sox2 negatively regulated the expression of some of the mTOR pathway such as 4EBP1, EIF4E and S6. FACS analysis in Sox2-transduced somatic cells also revealed a cell cycle arrest in G1-phase. Together our data suggest a role of Sox2 in cell fate and commitment.