Epithelial-mesenchymal Markers Research Articles

Artificial and Natural Sweeteners Biased T1R2/T1R3 Taste Receptors Transactivate Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition of Metastatic Phenotype.

Understanding the role of biased taste T1R2/T1R3 G protein-coupled receptors (GPCR) agonists on glycosylated receptor signaling may provide insights into the opposing effects mediated by artificial and natural sweeteners, particularly in cancer and metastasis. Sweetener-taste GPCRs can be activated by several active states involving either biased agonism, functional selectivity, or ligand-directed signaling. However, there are increasing arrays of sweetener ligands with different degrees of allosteric biased modulation that can vary dramatically in binding- and signaling-specific manners. Here, emerging evidence proposes the involvement of taste GPCRs in a biased GPCR signaling crosstalk involving matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu-1) activating glycosylated receptors by modifying sialic acids. The findings revealed that most natural and artificial sweeteners significantly activate Neu-1 sialidase in a dose-dependent fashion in RAW-Blue and PANC-1 cells. To confirm this biased GPCR signaling crosstalk, BIM-23127 (neuromedin B receptor inhibitor, MMP-9i (specific MMP-9 inhibitor), and oseltamivir phosphate (specific Neu-1 inhibitor) significantly block sweetener agonist-induced Neu-1 sialidase activity. To assess the effect of artificial and natural sweeteners on the key survival pathways critical for pancreatic cancer progression, we analyzed the expression of epithelial-mesenchymal markers, CD24, ADLH-1, E-cadherin, and N-cadherin in PANC-1 cells, and assess the cellular migration invasiveness in a scratch wound closure assay, and the tunneling nanotubes (TNTs) in staging the migratory intercellular communication. The artificial and natural sweeteners induced metastatic phenotype of PANC-1 pancreatic cancer cells to promote migratory intercellular communication and invasion. The sweeteners also induced the downstream NFκB activation using the secretory alkaline phosphatase (SEAP) assay. These findings elucidate a novel taste T1R2/T1R3 GPCR functional selectivity of a signaling platform in which sweeteners activate downstream signaling, contributing to tumorigenesis and metastasis via a proposed NFκB-induced epigenetic reprogramming modeling.

Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition Metastatic Phenotype.

Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have more than one active state, a phenomenon called either 'biased agonism', 'functional selectivity', or 'ligand-directed signaling'. However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called 'biased modulation', that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between neuraminidase-1 (Neu-1) and matrix metalloproteinase-9 (MMP-9) in the activation of glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Aravnil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue, PANC-1, and SW-620 cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link that regulates the interaction and signaling mechanism among these molecules present on the cell surface. Moreover, the study results demonstrate that CB1 agonists induce NFκB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial-mesenchymal markers, E-cadherin, and vimentin in SW-620 cells, albeit the impact on E-cadherin expression is less pronounced compared to vimentin. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis.

Correlation between E-cadherin/β-catenin, Vimentin expression, clinicopathologic features and drug resistance prediction in naïve prostate cancer: A molecular and clinical study.

Although epithelial-mesenchymal markers play an important role in prostate cancer (PC), further research is needed to better understand their utility in diagnosis, cancer progression prevention, and treatment resistance prediction. Our study included 111 PC patients who underwent transurethral resection, as well as 16 healthy controls. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to examine the expression of E-cadherin, β-catenin, and Vimentin. We found that E-cadherin and β-catenin were underexpressed in primary PC tissues. E-cadherin expression was found to be inversely associated with prostate-specific antigen progression (PSA-P; serum marker of progression; p = 0.01; |r| = 0.262). Furthermore, the underexpression of two markers, E-cadherin and β-catenin, was found to be associated with advanced tumor stage and grade (p < 0.05). On the other hand, Vimentin was overexpressed in PC patients with a fold change of 2.141, and it was associated with the diagnosis, prognosis, and prediction of treatment resistance to androgen deprivation therapy (p = 0.002), abiraterone-acid (p = 0.001), and taxanes (p = 0.029). Moreover, the current study highlighted that poor survival could be significantly found in patients who progressed after primary surgery, did not use drugs, and expressed these genes aberrantly. In Cox regression multivariate analysis (p < 0.05), a positive correlation between the Vimentin marker and coronary heart disease in PC patients was identified (p = 0.034). In summary, the present study highlights the diagnostic (p < 0.001), prognostic (p < 0.001), and therapeutic potential of Vimentin in primary PC (p < 0.05), as well as its implications for cardiovascular disease. Furthermore, we confirm the potential prognostic value of E-cadherin and β-catenin.

Forskolin affects proliferation, migration and Paclitaxel-mediated cytotoxicity in non-small-cell lung cancer cell lines via adenylyl cyclase/cAMP axis

Non-Small-Cell Lung Cancer (NSCLC) is considered one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Despite the undoubted therapeutic advances that have occurred in clinical practice over time, due to its high degree in both heterogeneity and resistance, NSCLC remains largely incurable. As a natural cAMP elevating agent, Forskolin has shown anti-cancer properties in different tumor types, thus supposing its possible usage in treating malignancies. In this study, we investigated the Forskolin outcome in H1299 and A549 NSCLC cell lines, either alone or in combination with Paclitaxel. We proved that Forskolin impairs cell growth and migration ability of these cells, concurrently. Albeit with a different extent between H1299 and A549, changes in cell-cycle progression and epithelial-mesenchymal markers were observed in response to Forskolin administration. Interestingly, comparable cell growth impairment was also obtained with the cAMP phosphodiesterase inhibitor IBMX, while the employment of adenylyl cyclase inhibitor SQ22536 counteracted, at least in part, the Forskolin-mediated anticancer effects. Besides as a single agent, we also demonstrated that Forskolin strongly enhances Paclitaxel-induced cytotoxicity, affecting cell death mainly via apoptosis induction. Notably, H89-mediated protein kinase A (PKA) inhibition further deteriorated the combination outcome. Altogether, our data designate Forskolin as a possible anticancer molecule in NSCLC, and recognize the adenylyl cyclase/cAMP axis as one of the pathways involved in. Although achieved at preclinical stage, our findings encourage the design of future studies aimed at further exploring the Forskolin employment in NSCLC treatment.

3-Bromopyruvate Suppresses the Malignant Phenotype of Vemurafenib-Resistant Melanoma Cells.

(1) BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells.

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial–mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells’ infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.

Hedgehog Signaling Pathway Orchestrates Human Lung Branching Morphogenesis.

The Hedgehog (HH) signaling pathway plays an essential role in mouse lung development. We hypothesize that the HH pathway is necessary for branching during human lung development and is impaired in pulmonary hypoplasia. Single-cell, bulk RNA-sequencing data, and human fetal lung tissues were analyzed to determine the spatiotemporal localization of HH pathway actors. Distal human lung segments were cultured in an air-liquid interface and treated with an SHH inhibitor (5E1) to determine the effect of HH inhibition on human lung branching, epithelial-mesenchymal markers, and associated signaling pathways in vitro. Our results showed an early and regulated expression of HH pathway components during human lung development. Inhibiting HH signaling caused a reduction in branching during development and dysregulated epithelial (SOX2, SOX9) and mesenchymal (ACTA2) progenitor markers. FGF and Wnt pathways were also disrupted upon HH inhibition. Finally, we demonstrated that HH signaling elements were downregulated in lung tissues of patients with a congenital diaphragmatic hernia (CDH). In this study, we show for the first time that HH signaling inhibition alters important genes and proteins required for proper branching of the human developing lung. Understanding the role of the HH pathway on human lung development could lead to the identification of novel therapeutic targets for childhood pulmonary diseases.

HH pathway orchestrates the epithelial‐mesenchymal crosstalk during human lung branching morphogenesis

RationaleHedgehog (HH) pathway plays an essential role in lung branching morphogenesis in rodents. However, little is known about the role of this pathway in human lung development. We hypothesize that HH pathway is necessary for branching during human lung development and hence in pulmonary hypoplasia.MethodsExpression of HH pathway components was analyzed in single cell and bulk RNA‐sequencing data from publicly available lung datasets. Fresh human fetal lung tissues between gestational ages 10‐18 weeks were collected. RT‐qPCR, fluorescent in situ hybridization (FISH) and immunofluorescence staining (IF) were performed to determine spatiotemporal localization of HH pathway actors. In addition, distal lung segments were cultured in air‐liquid interface for up to 48 hours in the presence or absence of HH inhibitor (5E1). RT‐qPCR, western blot and IF were performed to investigate the effect of HH pathway inhibition on epithelial‐mesenchymal markers and associated signaling pathways.ResultsBulk and scRNA‐seq analysis showed an early expression of HH pathway components during human lung development. We noted an inverse expression between activators and inhibitors receptors (PTCH1/HHIP) over time. PTCH1 had a higher expression at 20 weeks compared to 14 weeks while the opposite was observed for HHIP. HHIP FISH staining was restricted to ACTA2+ airway smooth muscle cells while PTCH1 was primarily localized in mesenchymal populations adjacent to the tip of airway epithelial buds where SHH ligand is produced. Inhibiting HH signaling ex vivo reduced branching during development (2.5‐fold vs control, p&lt;0.01, n=8). Consequently, SOX9expression, an epithelial progenitor marker, was increased whereas ACTA2 was decreased (respectively 1.5‐ and 2‐fold vs control, p&lt;0.05, n=8). These results were confirmed by western blot and IF staining. No change was observed for SOX2 gene expression while a significant increase in protein expression was observed. FGF10 increased following HH inhibition (1.5‐fold vs control, p&lt;0.01, n=8), but the expression of ETV4and 5 decreased (1.5‐fold vs control, p&lt;0.05, n=8). Finally, we demonstrated that HH signaling elements (including SHH, PTCH1, GLI2) were downregulated in lung tissues from congenital diaphragmatic hernia patients.ConclusionWe have determined for the first‐time gene expression and localization of HH pathway during human lung morphogenesis. Moreover, we showed that HH signaling inhibition alters important genes and proteins expression, such as epithelial‐mesenchymal progenitors and associated pathways, required for proper branching processes. Understanding the role of HH pathway on human lung development could lead to the identification of novel therapeutic targets for childhood pulmonary diseases.

Epithelial-mesenchymal transition related to bone invasion in oral squamous cell carcinoma

IntroductionBone invasion is an important prognostic factor in oral squamous cell carcinoma, leading to a lower survival rate and the use of aggressive treatment approaches. Epithelial-mesenchymal transition (EMT) is possibly involved in this process, because it is often related to mechanisms of cell motility and invasiveness. This study examined whether a panel of epithelial-mesenchymal markers are present in cases of oral squamous cell carcinoma with bone invasion and whether these proteins have any relationship with patients’ clinical-pathological parameters and prognostic factors. MethodsImmunohistochemical analysis of E-cadherin, twist, vimentin, TGFβ1, and periostin was performed in paraffin-embedded samples of 62 oral squamous cell carcinoma cases. ResultsThe analysis revealed that most cases (66%) presented with a dominant tumor infiltrative pattern in bone tissue, associated with lower survival rates, when compared with cases with a dominant erosive invasion pattern (P = 0.048). Twenty-seven cases (43%) expressed markers that were compatible with total or partial EMT at the tumor-bone interface. There was no association between evidence of total or partial EMT and other demographic or prognostic features. E-cadherin-positive cases were associated with tobacco smoking (P = 0.022); vimentin-positive cases correlated with tumors under 4 cm (P = 0.043). Twistexpression was observed in tumors with a dominant infiltrative pattern (P = 0.041) and was associated with the absence of periostin (P = 0.031). ConclusionWe observed evidence of total or partial EMT in oral squamous cell carcinoma bone invasion. The transcription factor twist appears to be involved in bone invasion and disease progression.

Paclitaxel resistance is mediated by NF-κB on mesenchymal primary breast cancer cells.

Paclitaxel has been used widely to treat breast cancer and other types of cancer. However, resistance is a major cause of failure for treatment and results in cancer progression. The present study investigated the association between paclitaxel resistance and the mesenchymal phenotype, using a model of primary breast cancer cells and employing four different cultures, two with an epithelial phenotype (MBCDF and MBCD17) and two with a mesenchymal phenotype (MBCDF-D5 and MBCD3). Epithelial-mesenchymal markers were evaluated by western blotting; MBCDF and MBCD17 cells expressed E-cadherin, SNAIL, Slug, and Twist, low levels of N-cadherin, but not vimentin. MBCDF-D5 and MBCD3 cells expressed N-cadherin, vimentin, and higher levels of SNAIL, and low levels of E-cadherin, Slug, and Twist. Cell viability was evaluated using a crystal violet assay after paclitaxel treatment; primary breast cancer cells with mesenchymal phenotype were resistant to paclitaxel compared with the epithelial primary breast cancer cells. Furthermore, using western blotting, it was revealed that mesenchymal cells had elevated levels of nuclear factor-κΒ (NF-κB) p65 and IκB kinase (IKK). Additionally, it was demonstrated that paclitaxel-induced degradation of the inhibitor of NF-κB, activation of NF-κB in a dose-dependent manner, and Bcl-2 and Bcl-xL upregulation. Finally, employing western blotting and crystal violet assays, the effects of the proteasome inhibitor ALLN were assessed. ALLN inhibited paclitaxel-induced NF-κB activation and restored the sensitivity to paclitaxel. Together, these data suggest that targeting the NF-κB/IKK axis might be a promising strategy to overcome paclitaxel resistance.

Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome.

Simple SummaryCirculating tumor cells (CTCs) detected in cancer patients as single cells or in clusters are of prognostic value. There is currently no information concerning the co-expression of mesenchymal markers (important for metastasis) and PD-L1 (immune system suppressor) in CTCs of metastatic breast cancer patients. In this study, we aimed to evaluate the co-expression of these markers in single and cluster CTCs and to determine if there is any relationship with patients’ outcome after eribulin treatment. CTCs were detected and phenotypically analyzed by quantifying keratins (epithelial marker), vimentin (mesenchymal marker) and PD-L1 using immunofluorescence and confocal microscopy. We found, for the first time, distinct CTC subpopulations, such as cluster and single PD-L1+ mesenchymal CTCs, at baseline and eight days after eribulin administration, respectively, that were associated with worse prognosis. Our study supports the role of CTC analysis in the investigation of mechanisms of resistance and disease progression in real time.We aimed to evaluate the co-expression of PD-L1 and epithelial-mesenchymal markers in CTCs from metastatic breast cancer (MBC) patients and to determine if there is any relationship with patients’ outcome after eribulin treatment. Using cytospin preparations of peripheral blood mononuclear cells (PBMCs) from MBC patients treated with eribulin and a combination of immunocytochemistry and immunofluorescence, we quantified PD-L1, keratins and vimentin in single and cluster CTCs on days 1 and 8 of the first-treatment cycle. CTCs (n = 173) were found in 31 out of 38 patients. At baseline, the presence of cluster CTCs (p = 0.048), cluster mesenchymal CTCs (mCTCs) (p = 0.0003) or cluster PD-L1+mCTCs (p = 0.006) was associated with shorter overall survival (OS). In multivariate cox regression analysis, the detection of cluster mCTCs was the only parameter associated with increased risk of death (p = 0.024). On day 8 post-eribulin administration, PD-L1+mCTCs and especially single PD-L1+mCTCs decreased in 75% and 89% of patients, respectively. The detection of single PD-L1+mCTCs after eribulin treatment was correlated with shorter PFS (p = 0.047) and OS (p = 0.020). In conclusion, our study identified for the first time that cluster and single PD-L1+mCTCs subpopulations are of clinical significance in patients with MBC and highlighted the importance of CTC phenotyping during treatment with eribulin.

P1.03-08 RNF43 Ubiquitinates and Degrades Phosphorylated E-Cadherin by c-Src to Facilitate Epithelial-Mesenchymal Transition in Lung Adenocarcinoma

In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). However, the precise mechanisms remain unclear. Protein antibody microarray was applied to identify the potential E3 ligase to downregulate E-cadherin. EMT was determined by morphology, epithelial-mesenchymal markers and aggressive behavior in vivo with the lung adenocarcinoma cell lines and tissues. The molecular biological methods including western blotting, immunoprecipitation, MALDI-Mass, immunohistochemistry, et al were applied to explore the signaling of RNF43-mediated EMT. Protein antibody microarray and E3 ligase profiling revealed that RING finger protein 43 (RNF43) is linked with E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, c-Src-Caspase-8 interaction markedly increased c-Src activity. Activated c-Src phosphorylated E-cadherin at the tyrosine 797 site to initiate RNF43-mediated E-cadherin ubiquitination at lysine 816 and subsequent degradation, thus freeing β-catenin into the nucleus to upregulate Vimentin and RNF43 in lung adenocarcinoma cell. Decreased E-cadherin and increased Vimentin induced EMT phenotype and promoted tumor metastasis. Frizzled 8 (Frz8)-RNF43 induced ubiquitination of phosphorylated E-cadherin was blocked by monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43. Our data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma, through ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.

Abstract 2011: Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy

Abstract Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy. Citation Format: Yi Yang, Woo Kyun Bae, Kyung Hwa Lee, Kyung-Sub Moon, So-Yeon Park, Rui Zhou, Isa Taş, Chathurika Gamage, Sueun Lee, Chaeyong Jung, Kyung Keun Kim, Hyung-Ho Ha, Hangun Kim. Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy [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 2011.

Ferritinophagic Flux Activation in CT26 Cells Contributed to EMT Inhibition Induced by a Novel Iron Chelator, DpdtpA.

Epithelial-mesenchymal transition (EMT) contributes to metastasis and drug resistance; inhibition of EMT may attenuate metastasis and drug resistance. It has been demonstrated that ferritinophagy involves the process of many diseases; however, the relationship between EMT and ferritinophagy was not fully established. Some iron chelators show the ability to inhibit EMT, but whether ferritinophagy plays a role in EMT is largely unknown. To this end, we investigated the effect of a novel iron chelator, DpdtpA (2,2 ′-di-pyridylketone dithiocarbamate propionic acid), on EMT in the CT26 cell line. The DpdtpA displayed excellent antitumor (IC50 = 1.5 ± 0.2 μM), leading to ROS production and apoptosis occurrence. Moreover, the ROS production correlated with ferritin degradation. The upregulation of LC3-II and NCOA4 from immunofluorescence and Western blotting analysis revealed that the occurrence of ferritinophagy contributed to ROS production. Furthermore, DpdtpA could induce an alteration both in morphology and in epithelial-mesenchymal markers, displaying significant EMT inhibition. The correlation analysis revealed that DpdtpA-induced ferritinophagy contributed to the EMT inhibition, implying that NCOA4 involved EMT process, which was firstly reported. To reinforce this concept, the ferritinophagic flux (NCOA4/ferritin) in either treated by TGF-β1 or combined with DpdtpA was determined. The results indicated that activating ferritinophagic flux would enhance ROS production which accordingly suppressed EMT or implementing the EMT suppression seemed to be through “fighting fire with fire” strategy. Taken together, our data demonstrated that ferritinophagic flux was a dominating driving force in EMT proceeding, and the new finding definitely will enrich our knowledge of ferritinophagy in EMT process.

Prognostic Role of Epithelial-Mesenchymal Transition Markers "E-Cadherin, β-Catenin, ZEB1, ZEB2 and p63" in Bladder Carcinoma.

BackgroundThis study aimed to investigate the expression of epithelial-mesenchymal markers’ E-cadherin, β-catenin, zinc-finger E-box-binding homeobox 1 (ZEB1), zinc-finger E-box-binding homeobox 2 (ZEB2) and p63 in transitional cell carcinoma (TCC) and squamous cell carcinoma (SCC) variants of bladder carcinoma (BC) and their correlation with clinicopathological parameters of prognostic importance.MethodsIn this retrospective study, 91 patients were enrolled (66 with TCC and 25 with SCC). All patients had full clinical and follow-up data and available paraffin blocks. Immunohistochemical analysis was performed and correlated with clinicopathological factors.ResultsIn TCC cases, reduced E-cadherin, β-catenin positivity and p63 expression rate were evident in the sitting of increased expression of ZEB1 and ZEB2. Patients with ZEB2 positive tumors were more likely to die compared to those with negative ZEB2 (P = 0.024). Moreover, in patients with muscle-invasive BCs, an intense p63 expression was associated with poor overall survival (OS) (P < 0.001). For patients with SCC, there was a reduction in E-cadherin and β-catenin positivity with elevated p63 expression and concomitant increased ZEB1 and ZEB2 expression. Poor prognosis was evident in association with reduced E-cadherin, positive nuclear β-catenin/reduced membranous β-catenin, ZEB1 and ZEB2 positive cases as well patients with elevated p63 expression (P < 0.001). TCC and SCC cases showed similar poor prognosis in association with elevated p63 expression (P < 0.001).ConclusionsIn both TCC and SCC variants, epithelial-mesenchymal transition (EMT) process is evident; however, its molecular mechanism shows some variations, specifically this notably different p63 expression pattern among two carcinoma variants with the similar impact of elevated p63 expression pattern on prognosis.

Potassium usnate, a water-soluble usnic acid salt, shows enhanced bioavailability and inhibits invasion and metastasis in colorectal cancer

Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy.

Targeting aberrant expression of Notch-1 in ALDH+ cancer stem cells in breast cancer.

We have previously reported that high aldehyde dehydrogenase (ALDH) enzyme activity in breast cancer cells results in breast cancer stem cell (BCSC) properties by upregualting Notch-1 and epithelial mesenchymal markers. This results in chemoresistance in breast cancer. Here, we examined the functional and clinical significance of ALDH expression by measuring the ALDH levels in breast cancer tissues by immunohistochemistry. There was a significantly higher ALDH expression in higher grade breast cancer tumor tissues (Grade- II and III) versus normal breast tissues. Injection of BCSC (ALDH+ and CD44+ /CD22- ) cells resulted in aggressive tumor growth in athymic mice versus ALDH- cells. The ALDH+ and CD44+ /CD22- tumors grow rapidly and are larger than ALDH- tumors which were slow growing and smaller. Molecularly, ALDH+ tumors expressed higher expression of Notch-1 and EMT markers than ALDH- tumors. Oral administration of the naturally occurring Psoralidin (Pso, 25 mg/kg of body weight) significantly inhibited the growth in ALDH+ and ALDH- tumors as well. Psoralidin inhibited Notch-1 mediated EMT activation in ALDH+ and ALDH- tumors-this confirms our in vitro findings. Our results suggest that Notch-1 could be an attractive target and inhibition of Notch-1 by Psoralidin may prevent pathogenesis of breast cancer as well as metastasis. © 2016 Wiley Periodicals, Inc.

Abstract 4953: Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer

Abstract Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs. Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. 189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5 to 10 cells. 17/34 (50%) CTC samples (4 patients) had shared sSNV with the paired tumor sample (range 0.35%-68%). Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients. We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented. Citation Format: Vincent Faugeroux, Céline Lefebvre, Emma Pailler, Valérie Pierron, Fanny Billiot, Charles Marcaillou, Philippe Vielh, Semih Dogan, Philippe Rameau, Maud Ngocamus, Jean Charles Soria, Karim Fizazi, Yohann Loriot, Sylvia Julien, Françoise Farace. Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4953.

Onco-miR-130 promotes cell proliferation and migration by targeting TGFβR2 in gastric cancer.

MicroRNAs (miRNAs) have been proved to play crucial roles in tumorigenesis. TGFβ signal pathway abnormality is found in various cancers and correlates with tumor proliferation and metastasis. However, the mechanisms underlying the dys-regulation of TGFβR2 expression in GC have not been investigated yet. In this study, we found that the TGFβR2 protein was clearly repressed in tumor tissues, while miR-130 expression level was dramatically increased in GC tissues. Firefly luciferase activity assay revealed that miR-130 could directly bind to 3′UTR of TGFβR2 mRNA. Meanwhile, miR-130 mimics lead to the decreased TGFβR2 protein levels, while miR-130 inhibitors enhanced TGFβR2 expression in SGC7901 cells. Subsequent functional experiments showed that overexpressed miR-130 could promote proliferation and migration of SGC7901 cells. And siRNA-mediated TGFβR2 down-regulation could simulate the effects of miR-130 mimics on phenotypes of SGC7901 cells. Furthermore, there existed intense relationship between the expression level of miR-130 and epithelial-mesenchymal markers. Our results demonstrated that miR-130 was an oncogene by directly targeting TGFβR2 in GC.

Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a KrasG12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. KrasG12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.