Epithelial-mesenchymal Transition Changes Research Articles

The prognostic impact of programmed cell death 1 and its ligand and the correlation with epithelial-mesenchymal transition in thymic carcinoma.

BackgroundThe significance of epithelial‐mesenchymal transition (EMT) and immune checkpoint proteins in thymic carcinoma remains unknown. We examined the clinical significance of EMT, tumor‐infiltrating lymphocytes expressing the immune checkpoint protein, programmed cell death 1 (PD‐1 + TILs), and the expression of PD‐1 ligand 1 (PD‐L1) in thymic carcinoma (TC). We also investigated the relationships between these immune checkpoint proteins and the EMT status and examined the impact of induction chemotherapy on patients with tumors that express these proteins.MethodsThe relationship between PD‐1 + TILs/PD‐L1 and clinicopathological findings including EMT was investigated by immunohistochemistry (IHC) of surgically resected samples from 43 patients with TC. In 15 patients receiving induction therapy (IT), those factors were compared before and after IT.ResultsWith IHC, 26 cases (60.5%) were positive for PD‐L1, and 19 cases were positive for PD‐1 + TILs (44.2%). The disease‐free survival rate in patients showing EMT and who were PD‐1/PD‐L1 positive was significantly worse compared to negative cases (EMT; P = 0.0095, PD‐1; P = 0.001, PD‐L1; P = 0.0037). We found a significant relationship between PD‐L1 and EMT status (P = 0.01). In patients who received IT, PD‐L1 increased, and the change was strongly correlated with EMT status (P = 0.01).ConclusionEpithelial‐mesenchymal transition, PD‐L1, and PD‐1 + TILs have prognostic impact, and PD‐L1 is correlated with EMT status. PD‐L1 expression after IT was significantly higher compared to before IT and was correlated with the EMT change. Thus, PD‐L1 may be upregulated during EMT, and anti‐PD‐1/PD‐L1 immunotherapy may provide reliable treatment of TC in combination with chemotherapy.

Alpha-lipoic acid suppresses Nε-(carboxymethyl) lysine-induced epithelial mesenchymal transition in HK-2 human renal proximal tubule cells

Nε-(carboxymethyl) lysine (CML) plays causal roles in diabetic complications. In the present study, we investigated whether CML-induced HIF-1α accumulation and epithelial-mesenchymal transition (EMT) in HK-2 renal proximal tubular epithelial cells. Treatment with CML-BSA increased reactive oxygen species (ROS) production reduced the mitochondrial membrane potential and induced mitochondrial fragmentation. Pre-treatment of cells with antioxidant, α-lipoic acid, normalised the ROS production and restored the mitochondrial membrane potential. These changes were accompanied with morphological changes of epithelial mesenchymal transition. CML-BSA increased the protein level of hypoxia-inducible factor-1α (HIF-1α), and the EMT-associated transcription factor, TWIST. These effects were reversed by α-lipoic acid. CML-BSA increased the protein levels of mesenchymal-specific markers, including vimentin, α-smooth muscle actin, which were alleviated by pre-treatment with α-lipoic acid. Our data suggest that CML-BSA induces EMT through a ROS/HIF-1α/TWIST-dependent mechanism, and that α-lipoic acid may alleviate the CML-induced EMT in renal tubular cells.

Epithelial-to-Mesenchymal Transition (EMT) and Drug Response in Dynamic Bioengineered Lung Cancer Microenvironment.

Tumor microenvironment and the interplay of physical and mechanical forces are key determinants of cancer initiation, progression, and response to drug treatment. However, the impact of tumor microenvironment on cancer progression is poorly understood, in large due to the lack of in vitro models that recapitulate the physical aspects of tumor microenvironment. Herein, a simple, dynamic 3D nonsmall cell lung carcinoma culture using a multichannel microfluidic model platform is developed for evaluating the contribution of flow-induced hydrodynamic shear stress on epithelial-to-mesenchymal transition (EMT). It is found that flow induces changes in cellular morphology and EMT in 2D and 3D when lung cancer A549 cells are cultured on a microfluidic chip under laminar flow for 4-5 days compared to traditional static cultures. The role of dynamic cell culture on chemotherapeutic effects is monitored. Drug response with an existing anti-cancer drug, e.g., erlotinib and an investigational drug (NSC-750212), shows distinct cytotoxic effects in flow compared to static cultures, suggesting a potential influence of flow on drug efficacy in 2D and 3D models. The platform demonstrates the ability to create a dynamic microscale tumor model, which could be explored as a tool for early drug screening and treatment monitoring in cancer and other diseases.

Gremlin Regulates Tubular Epithelial to Mesenchymal Transition via VEGFR2: Potential Role in Renal Fibrosis.

Chronic kidney disease (CKD) is emerging as an important health problem due to the increase number of CKD patients and the absence of an effective curative treatment. Gremlin has been proposed as a novel therapeutic target for renal inflammatory diseases, acting via Vascular Endothelial Growth Factor Receptor-2 (VEGFR2). Although many evidences suggest that Gremlin could regulate renal fibrosis, the receptor involved has not been yet clarified. Gremlin, as other TGF-β superfamily members, regulates tubular epithelial to mesenchymal transition (EMT) and, therefore, could contribute to renal fibrosis. In cultured tubular epithelial cells Gremlin binding to VEGFR2 is linked to proinflammatory responses. Now, we have found out that in these cells VEGFR2 is also involved in the profibrotic actions of Gremlin. VEGFR2 blockade by a pharmacological kinase inhibitor or gene silencing diminished Gremlin-mediated gene upregulation of profibrotic factors and restored changes in EMT-related genes. Moreover, VEGFR2 inhibition blocked EMT phenotypic changes and dampened the rate of wound healing in response to Gremlin. The role of VEGFR2 in experimental fibrosis was evaluated in experimental unilateral ureteral obstruction. VEFGR2 inhibition diminished the upregulation of profibrotic genes and EMT changes, as well as the accumulation of extracellular matrix proteins, such as fibronectin and collagens in the obstructed kidneys. Notch pathway activation participates in renal damage progression by regulating cell growth/proliferation, regeneration and inflammation. In cultured tubular epithelial cells, Notch inhibition markedly downregulated Gremlin-induced EMT changes and wound healing speed. These results show that Gremlin regulates the EMT process via VEGFR2 and Notch pathway activation, suggesting that the Gremlin/VEGFR2 axis could be a potential therapeutic target for CKD.

PIWI-like protein, HIWI2: A novel player in proliferative diabetic retinopathy

Diabetic retinopathy (DR) is one of the major causes of blindness resulting from prolonged hyperglycemia which leads to breakdown of blood retinal barrier and excessive neovascularization. In our previous study, we demonstrated the presence of germline-specific PIWI-like proteins in human retina and retinal pigment epithelium (RPE) and a discrete function of HIWI2 (PIWIL4) in the assembly of tight junction through Akt/GSK3α/β. Recently, PIWI/piRNA has been suggested to be involved in the development of diabetes. Here, we have investigated the role of HIWI2 in proliferative diabetic retinopathy (PDR). Interestingly, Western blot analysis indicated the elevated expression of HIWI2 in vitreous aspirates of patients with PDR in comparison to macular hole (MH) and rhegmatogenous retinal detachment (RRD). In addition, treatment of ARPE19 with 25% of PDR vitreous aspirate significantly increased the expression of HIWI2. Moreover, exposure of ARPE19 to oxidative stress and VEGF, induced the expression of HIWI2. Further, we knocked down HIWI2 in ARPE19 cells to understand its role in the disease progression. Silencing HIWI2 reduced the expression of growth factors, VEGF and TGFβ1, and altered the expression of epithelial to mesenchymal transition (EMT) markers E-cadherin and αSMA. In addition, expression of MMP9 and cell migration was reduced in Si-HIWI2. Collectively, our report highlights a novel function and association of a piRNA binding protein, HIWI2 to PDR. The elevated expression of HIWI2 in PDR could influence various aspects of the disease pathogenesis, like EMT changes and cell migration. Hence, understanding the exact function of HIWI2 in retina could reveal its potential as a therapeutic target for retinopathy.

Role of MAPK signaling pathway in epithelial-mesenchymal transition of type II alveolar epithelial cells induced by Paraquat

Objective: To investigate the roles of p38 mitogen-activated protein kinases (p38 MAPK) , extracellular regulated protein kinases (ERK) and c-Jun N-tenninal kinases (JNK) of MAPK signaling pathway in Paraquat-induced epithelial to mesenchymal transition (EMT) of type II alveolarepithelial cells. Methods: RLE-6NT cells were incubated with different concentrations of PQ (0, 25, 50, 100μmol/L) for 6, 12 and 24 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined using an MTT assay. Cell migration ability was detected using scratch wound assay. Protein expression of P-p38 MAP, P-Erk1/2, P-JNK, E-cad, ZO-1, Vimentin and а-SMA were detected by western blot. The level of genes related to fibrosis (COL-I, COL-III, FN and FSP-1) were analyzed via quantitative real-time RT-PCR. Results: Cell morphology started to undergo EMT changes with a phenotype characteristic of mesenchymal cells, including an elongated shape and a lack of tight cell-cell adhesions induced by 100μmol/L PQ treatment in a time-dependent manner. MTT showed that cell viability decreased with increasing PQ concentration (50、100、200、300 μmol/L PQ treatment for 24 h) and increasing treatment time (200 μmol/L PQ treatment for 6, 12, 24, 36, 48 h) . Compared to control group, the expressions of the epithelial phenotype marker E-cad and ZO-1 significantly decreased with PQ treatment (50, 100μmol/L) in a time-dependent manner (P<0.05) . Additionally, the level of the mesenchymal marker (a-SMA, vimentin) dramatically increased with PQ treatment in the same concentration-and time-dependent manner (P<0.05) . Cell migration ability was markedly increased after 24 h of 100 μmol/L PQ treatment compared to control (P<0.05) . The phosphorylated forms of p38 MAPK, Erk1/2, and JNK were increased at 24 h after stimulation with PQ (P<0.05) . This PQ induced (100 μmol/L) phosphorylation was markedly attenuated in the presence of the p38 MAPK, ERK and JNK inhibitors (SB-203580, SP-600125 and PD98059) respectively. Furthermore, RT-PCR showed that PQ significantly induced the upregulation expression of COL I and III mRNA, Fn, and FSP-1 mRNA (P<0.05) . Conclusion: PQ-induced pulmonary fibrosis occurs via EMT, which is mediated by the MAPK pathway.

MicroRNA-29b as a Potentially Therapeutic Molecular Via Binding to SP-1 in Silica Induced EMT and Pulmonary Fibrosis

Workers expose to crystalline silica dust could develop into silicotic fibrosis, which is characterized by diffused lung fibrosis and silicon nodules. Up to now, scholars have verified that epithelial-mesenchymal transition (EMT) is an important source of fibroblasts. During the process of EMT, epithelial and mesenchymal markers change significantly, in which transcription factors also play a vital role. Emerging evidence illustrates that microRNA-29b (miR-29b), which is one type of the non-coding small RNAs, mediates various pathogenic organ fibrotic processes by regulating the target gene transcriptionally. However, the function and mechanism of miR-29b that participates in silica-exposed pulmonary fibrosis remain unclear. In the present study, we found that silica induced EMT and pulmonary fibrosis, and the expression of miR-29b was downregulated significantly. This was performed not only in mice with silica induced EMT and MLE-12 cells treated with silica but also in the serum from workers, who had been diagnosed with the silicosis (phase I and II). In silica-induced EMT and lung fibrosis, the changes of epithelial markers (ZO-1, SP-C) and mesenchymal markers (a-SMA) were identified by RT-qPCR or Western blot, and the increasing expression of SP-1 and CTGF were also observed. Next, we fully illustrated that the down-regulation of miR-29b was associated with the up-regulation of SP-1 and CTGF by using luciferase assays. Further experiments verified that the overexpression of miR-29b inhibited silica-induced EMT in vitro. Moreover, the agomiR-29b blocked silica-induced EMT and regulated the levels of SP-1 and CTGF in mice. Our study elucidates that miR-29b have a therapeutic effect on silica induced EMT and lung fibrosis via mediation of SP-1. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No. 81273047, 91643114, 81472958, 81703197 and 81602832) and Key Projects of Science and Technology Program by Beijing Municipal Education Commission (No. KZ201610025020). Declaration of Interests: The authors declare that there are no conflicts of interest. Ethics Approval Statement: The research program was approved by the Research Ethics Committee of Capital Medical University. All protocols were completed according to approved guidelines.

Low-dose eribulin mesylate exerts antitumor effects in gastric cancer by inhibiting fibrosis via the suppression of epithelial&amp;ndash;mesenchymal transition and acts synergistically with 5-fluorouracil

BackgroundCharacterized by aggressive proliferation, extensive stromal fibrosis, and resulting drug resistance, peritoneal dissemination in gastric cancer remains associated with poor prognosis. Interaction between cancer and stromal cells accelerates tumor progression via epithelial–mesenchymal transition (EMT), which is one of the major causes of tissue fibrosis, and human peritoneal mesothelial cells (HPMCs) play important roles as cancer stroma in peritoneal dissemination. Transforming growth factor-β (TGF-β) has a pivotal function in the progression of EMT, and Smad proteins play an important role in the TGF-β signaling pathway. Eribulin mesylate (eribulin), a nontaxane microtubule dynamics inhibitor used for the treatment of advanced breast cancer, inhibits EMT changes in triple-negative breast cancer cells. We examined its ability to inhibit tumor progression and EMT changes resulting from the interaction between gastric cancer cells and HPMCs and to act synergistically with 5-fluorouracil (5-FU), a key drug for gastric cancer.Materials and methodsProliferation of gastric cancer cells and HPMCs isolated from healthy omentum was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Following gastric cancer cell/HPMC coculture, EMT markers were detected by immunofluorescence, immunohistochemistry, and Western blotting; invasion assays were performed; and TGF-β and Smad phosphorylation were assessed by Western blotting and enzyme-linked immunosorbent assay. A mouse fibrotic tumor xenograft model was established using gastric cancer cell/HPMC cocultures. The effect of eribulin and/or 5-FU was tested in each case.ResultsEribulin significantly suppressed gastric cancer cell proliferation and EMT changes in MKN-45 gastric cancer cells and HPMCs induced by their interaction in vitro. Eribulin inhibited EMT at much lower concentrations (≥0.5 nM for MKN-45 and ≥0.1 nM for HPMCs) than its half maximal inhibitory concentrations (2.2 nM for MKN-45 and 8.1 nM for HPMCs), and this resulted, at least partly, from the downregulation of TGF-β/Smad signaling. Eribulin administration of ≥0.1 mg/kg suppressed tumor progression (0.1 mg/kg, p=0.02), and fibrosis was inhibited by lower dose (0.05 mg/kg, p=0.008) in the xenograft model. Furthermore, 0.05 mg/kg administration with 5-FU brought about synergistic antitumor effects (p=0.006).ConclusionLow-dose eribulin combined with 5-FU might be a promising therapy for peritoneal dissemination in gastric cancer.

Role of histone deacetylase 1 in distant metastasis of pancreatic ductal cancer.

Current therapies for pancreatic ductal cancer (PDAC) do not sufficiently control distant metastasis. Thus, new therapeutic targets are urgently needed. Numerous studies have suggested that the epithelial‐mesenchymal transition (EMT) is pivotal for metastasis of carcinomas. The fact that the EMT is reversible suggests the possibility that it is induced by an epigenetic mechanism. In this study, we aimed to investigate the role of histone deacetylase 1 (HDAC1), which is an epigenetic mechanism on distant metastasis of PDAC. We investigated the HDAC1 expression in 103 resected PDAC specimens obtained from patients who were treated with/without preoperative therapy using immunohistochemistry. To validate the findings in the clinical samples, we evaluated the HDAC1 activity, the EMT‐associated genes and the migration/invasion ability in vitro, and performed an HDAC1 inhibitor assay. The high expression of HDAC1 in clinical samples was significantly associated with poor progression‐free survival, especially distant metastasis‐free survival. In vitro, HDAC1 inhibitors decreased the invasion ability and reversed the EMT change; the only factor to show a concomitant decrease was the expression of SNAIL. We confirmed that the HDAC1 expression was associated with the SNAIL expression in clinical samples. Moreover, the resistant cells and parental cells did not show any significant differences in the expression of HDAC1; this was consistent with the finding that preoperative therapy did not alter the HDAC1 expression in clinical samples. The targeting of HDAC1, which could suppress metastasis by inhibiting the EMT, is a promising treatment option for PDAC.

Aldose Reductase Inhibition Prevents Development of Posterior Capsular Opacification in an In Vivo Model of Cataract Surgery.

PurposeCataract surgery is a procedure by which the lens fiber cell mass is removed from its capsular bag and replaced with a synthetic intraocular lens. Postoperatively, remnant lens epithelial cells can undergo an aberrant wound healing response characterized by an epithelial-to-mesenchymal transition (EMT), leading to posterior capsular opacification (PCO). Aldose reductase (AR) inhibition has been shown to decrease EMT markers in cell culture models. In this study, we aim to demonstrate that AR inhibition can attenuate induction of EMT markers in an in vivo model of cataract surgery.MethodsA modified extracapsular lens extraction (ECLE) was performed on C57BL/6 wildtype, AR overexpression (AR-Tg), and AR knockout mice. Immunofluorescent staining for the myofibroblast marker α-smooth muscle actin (α-SMA), epithelial marker E-cadherin, and lens fiber cell markers αA-crystallin and Aquaporin 0 was used to characterize postoperative PCO. Quantitative reverse transcription PCR (qRT-PCR) was employed to quantify postoperative changes in α-SMA, vimentin, fibronectin, and E-cadherin. In a separate experiment, the AR inhibitor Sorbinil was applied postoperatively and qRT-PCR was used to assess changes in EMT markers.ResultsGenetic AR knockout reduced ECLE-induced upregulation of α-SMA and downregulation of E-cadherin. These immunofluorescent changes were mirrored quantitatively in changes in mRNA levels. Similarly, Sorbinil blocked characteristic postoperative EMT changes in AR-Tg mice. Interestingly, genetic AR knockout did not prevent postoperative induction of the lens fiber cell markers αA-crystallin and Aquaporin 0.ConclusionsAR inhibition prevents the postoperative changes in EMT markers characteristic of PCO yet preserves the postoperative induction of lens fiber cell markers.

Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial-mesenchymal transition, invasion, and migration.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease with poor prognosis. Because of early metastasis prior to diagnosis and therapeutic resistance, ESCC has become one of the leading causes of cancer‐related death. Here, we investigated the clinicopathological significance of the association of octamer‐binding transcription factor 4 (OCT4) with lymphoid enhancer‐binding factor 1 (LEF1) expression and the potential molecular mechanism in the epithelial‐mesenchymal transition (EMT), invasion, and migration of ESCC. The expression of OCT4 and LEF1 was detected via immunohistochemistry analysis. High levels of LEF1 expression were observed in 95 ESCC specimens and were obviously associated with aberrant clinicopathological features and poor patient prognosis. Our previous study showed that OCT4 expression level is elevated in ESCC, and statistical analysis showed that the elevated expression of OCT4 and LEF1 in ESCC was significantly associated with histologic grade, lymph node metastasis, TNM stage, and poor patient prognosis. The specific inhibition of OCT4 expression via a lentivirus encoding OCT4‐shRNA (LV‐shOCT4) in Eca109 cells led to decreased levels of OCT4 and LEF1 in vitro. Additionally, we applied a rescue strategy by infecting LV‐shOCT4 Eca109 cells with a LEF1 overexpression plasmid (p‐LEF1) and detected changes in EMT, migration, and invasion. Unsurprisingly, the p‐LEF1 group exhibited greater EMT, invasion, and migration than did the LV‐shOCT4 and negative control groups. This study demonstrates for the first time the relationship between OCT4 and LEF1 expression. The combination of high expression of OCT4 and LEF1 was associated with clinicopathological features of atypical patients, and this combination might be an ideal prognostic factor in ESCC. OCT4 positively regulated LEF1 expression, and LEF1 mediated the effects of OCT4 in cancer cell EMT, invasion, and migration. The data presented here suggest that the inhibition of OCT4‐LEF1 signaling may be a new therapeutic target for the treatment of ESCC.

Abstract 3960: The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes

Abstract Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression. The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes. Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed. Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network. Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors. Citation Format: Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3960.

Metformin suppresses the invasive ability of pancreatic cancer cells by blocking autocrine TGF‑β1 signaling

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive neoplasm with a 5‑year survival rate of <8%. Metformin, the most widely used antidiabetic drug in the world, has been shown to exert anticancer activities in epidemiological and animal studies. Our previous studies revealed that metformin suppressed desmoplasia in PDAC by reducing TGF‑β1 production in cancer cells. The aim of the present study was to investigate the effects of metformin on invasion and epithelial‑mesenchymal transition (EMT) in pancreatic cancer and to reveal the underlying mechanisms. In the present study, we revealed that metformin suppressed migration, invasion and EMT changes in pancreatic cancer cells. Furthermore, metformin reduced TGF‑β1 production and Smad2/3 phosphorylation in pancreatic cancer cells. In addition, treatment with recombinant TGF‑β1 recovered the metformin‑mediated invasion inhibition and EMT changes. Treatment with metformin also suppressed tumor growth, invasion and EMT in LSL‑KrasG12D/+, Trp53fl/+and Pdx1‑Cre (KPC) transgenic mice that harbor spontaneous pancreatic cancer. Collectively, our study revealed a new possible mechanism for the antitumor effects of metformin via autocrine TGF‑β1/Smad2/3 signaling in PDAC.

Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer.

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non–small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib)-resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.

P38 MAPK is Crucial for Wnt1- and LiCl-Induced Epithelial Mesenchymal Transition.

Idiopathic pulmonary fibrosis (IPF) is characterized by myofibroblast foci in lung parenchyma. Myofibroblasts are thought to originate from epithelial-to-mesenchymal transition (EMT). Wnt1 and lithium chloride (LiCl) induce EMT in alveolar epithelial cells (AECs), but the mechanisms are unclear. AECs were treated with Wnt1 and LiCl, respectively; morphological change and molecular changes of EMT, including E-cadherin, fibronectin, and vimentin, were observed. SB203580 was administrated to test the role of p38 МАРК signaling in EMT. Then AECs were treated with siRNAs targeting p38 МАРК to further test the effects of p38 МАРК, and the role was further confirmed by re-expression of p38 МАРК. At last P-catenin siRNA was used to test the role of β-catenin in the EMT process and relationship of β-catenin and p38 МАРК was concluded. Exposure of AECs to Wnt1 and LiCl resulted in upregulation of vimentin and fibronectin with subsequent downregulation of E-cadherin. Wnt1 and LiCl stimulated the p38 МАРК signaling pathways. Perturbing the p38 МАРК pathway either by SB203580 or through p38 МАРК siRNA blocked EMT and inhibited fibronetin synthesis, which were reversed by transfection of p38 МАРК expression plasmid. β-catenin siRNA attenuated the EMT process and decreased p38 МАРК phosphorylation, indicating that β-catenin is involved in the EMTrelated changes through regulation of p38 МАРК phosphorylation. These findings suggest that p38 МАРК participates in the pathogenesis of EMT through Wnt pathway and that p38 МАРК may be a novel target for IPF therapy.

Gestational low-protein intake enhances whole-kidney miR-192 and miR-200 family expression and epithelial-to-mesenchymal transition in rat adult male offspring.

Studies have shown that adult offspring of mothers fed a protein-restricted diet during pregnancy present a pronounced reduction of nephron number associated with decreased fractional urinary sodium excretion and arterial hypertension. Additionally, recent advances in our understanding of the molecular pathways that govern the association of gestational nutritional restriction, intrauterine growth retardation and inflammation with impaired nephrogenesis, nephron underdosing and kidney fibrosis point to the epithelial to mesenchymal transition (EMT) as a common factor. In the current study, protein and sodium urinary excretion rates were evaluated in rats, and immunohistochemistry and western blot techniques were used to characterize kidney structure changes in 16week old male offspring of mothers fed a low-protein diet during pregnancy (LP group) compared with age-matched (NP) controls. We also verified the expression of miRNA, mRNA and protein markers of fibrosis and the EMT in whole kidney prepared from LP offspring. We found, surprisingly, that arterial hypertension and long-term hyperfiltration, manifest by proteinuria, were associated with increased renal miR-192 and miR-200 family expression in 16week old LP relative to age-matched NP rats. Measurement of kidney fibrosis and EMT-related protein markers, by histochemistry and immunoblot techniques, showed a significant rise of TGF-β1 and type-I collagen content in glomeruli and tubulointerstitial areas, accompanied by enhanced fibronectin and ZEB1 and decreased E-cadherin immunoreactivity in 16week old LP offspring. The results were partially confirmed by increased gene (mRNA) expression of collagen 1α1, collagen 1α2 and ZEB1 in LP whole kidneys compared with those of age-matched NP offspring. In view of the presumed functional overload in the remaining nephrons, we suggest that hypertension and proteinuria development following maternal protein restriction may be a preponderant factor for EMT and structural kidney changes in LP offspring. However, our study was not wholly able to establish the precise role of miRNAs in LP kidney disorders. Thus, further studies will be required to assess the contribution of the miR family to renal injury in a gestational protein-restricted model of fetal programming.

CAFs enhance paclitaxel resistance by inducing EMT through the IL‑6/JAK2/STAT3 pathway.

Carcinoma-associated fibroblasts (CAFs) are the major components of mesenchymal cells in the inflammatory tumor microenvironment. They are involved in epithelial-mesenchymal transition (EMT) and chemotherapy resistance by directly contacting with cancer cells or secretory cytokines. In the present study, we examined the role of CAFs in the induction of EMT in ovarian cancer. Primary ovarian cancer cells, CAFs and normal fibroblasts (NFs) were isolated from fresh cancer tissue and cultured for immunohistochemistry studies. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-6 in the culture supernatants of these cells. The expression of IL-6 at the mRNA level was examined by RT-PCR. The expression of IL-6 at the protein level in ovarian cancer tissues was determined using an immunofluorescence assay in both tissue sections and cell lobes. OVCAR3 cells were treated with the culture supernatants collected from CAFs and NFs. IL-6 monoclonal antibody (mAb) was employed to neutralize IL-6. The expression of phosphorylated STAT3 was assessed. Changes in EMT, proliferation, invasion and proapoptotic protein expression were also examined. Flow cytometry was performed to detect the changes in apoptosis resistance of OVCAR3 cells. The JAK2/STAT3 pathway-specific inhibitor AG490 was used to block this pathway and the β-TGF inhibitor was used to inhibit EMT. The clinical data of patients treated in our hospital were collected between January 1st, 2009 and June 30th, 2013. The expression of interstitial IL-6 in paraffin-embedded tissues was detected by immunohistochemistry. The relationship between the expression of interstitial IL-6 and the treatment response was examined by linear regression and multiple linear regression analyses. We found that CAFs were the main source of IL-6 in ovarian cancer tissue. CAFs promoted the phosphorylation of STAT3 in ovarian cancer and enhanced the proliferation, invasion and EMT. Enhanced EMT may lead to apoptosis resistance, inhibitory expression of pro-apoptotic proteins and paclitaxel resistance. A total of 255 patients were enrolled in this retrospective study. Univariate and multivariate analyses revealed that age, CA125, interstitial IL-6 expression and cytoreduction satisfaction were closely related to the sensitivity of the TP (docetaxel plus cisplatin or carbopatin) regimen in ovarian cancer (P<0.05). These results demonstrated that CAFs highly secreted IL-6 and promoted β-TGF-mediated EMT in ovarian cancer via the JAK2/STAT3 pathway, leading to inhibited apoptosis and subsequent paclitaxel resistance. Therefore, CAFs may be a new therapeutic target for the treatment of ovarian cancer.

Abstract P5-05-05: Patient-derived triple negative breast cancer xenografts as a translational model to screen for novel kinase pathways

Abstract Overall, triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype, which has higher rates of metastasis and recurrence. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2, c-FOS, SNAI1, ZEB1) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Matossian M, Burks H, Bowles A, Sabol R, Hoang V, Elliott S, Bunnell B, Zuercher W, Drewry D, Wells C, Alfortish A, Lee S, Hartono A, Jones S, Moroz K, Zea A, Burow M, Collins-Burow B. Patient-derived triple negative breast cancer xenografts as a translational model to screen for novel kinase pathways [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 P5-05-05.

Abstract P4-14-02: CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) accounts for 2% of breast cancers but 10% of breast cancer-related deaths in the US. Clinical hallmarks of IBC are tumor cell emboli in lymphatic vessels and overexpression of E-cadherin, which promotes cell clustering. Given these hallmarks, IBC is thought to spread via collective invasion and cell clusters. However, we showed that IBC cells underwent epithelial-to-mesenchymal transition (EMT) and metastasized through EMT. Thus, there are two contradictory theories of IBC metastasis. The objectives of this study were 1) to propose a model that reconciles these two models, and 2) to identify target molecules for inhibition of IBC metastasis. Methods: We previously showed that Matrigel culture induced EMT-like changes in SUM149 IBC cells. To test if this transformation from epithelial (E) to mesenchymal (M) in Matrigel culture is unique to IBC cells, a panel of breast cancer cells was cultured in both monolayer and Matrigel-coated plates. The cells were IBC (SUM149, SUM190, KPL4, IBC3), triple-negative breast cancer (TNBC) (MDA-MB-231, MDA-MB-468), and ER+ (MCF7) cells. Phenotypic changes in morphology and expression of EMT markers (E-cadherin, vimentin) were captured with bright field and immunofluorescent (IF) images, respectively. For genome-wide and targeted transcriptional analysis, SUM149 cells cultured in monolayer and Matrigel were processed using DNA microarrays and Taqman qRT-PCR. To correlate the Matrigel gene signature with M features in human breast cancer, a human breast cancer data set was hierarchically clustered with the Matrigel gene signature. Results: SUM149 cells showed a remarkable phenotypic change from E in monolayer culture to M in Matrigel. IF analysis confirmed induction of vimentin expression in Matrigel but stable expression of E-cadherin (thus, we refer to this state as E/M hybrid). This trend was also observed with SUM190 cells. Using qRT-PCR, we confirmed downregulation of E-cadherin and upregulation of M markers (vimentin, Twist1, Snail1, ZEB2) in Matrigel-cultured SUM149 and SUM190 cells compared to monolayer-cultured cells. DNA microarray transcriptional analysis confirmed this trend in SUM149 cells. TNBC has more M-like features than other breast cancer subtypes. Given this evidence, we clustered human breast cancer data using overexpressed genes in Matrigel-cultured SUM149 cells. We identified a cluster of 20 genes in TNBC samples and, assuming that these genes are drivers of E to E/M transition, chose the inflammation-related gene CSF1 as a candidate. The CSF1/CSF1R axis was inhibited by a CSF1R inhibitor, BLZ945; moreover, treatment with BLZ945 reversed the EMT changes in cells in Matrigel culture. Treatment with 5 μM BLZ945 re-induced E-cadherin expression and suppressed Snail1 and Twist1 expression in Matrigel-cultured SUM149 cells. Conclusion: IBC cells are more prone to undergo transition from E to E/M phenotype in Matrigel culture than are cells of other breast cancer subtypes. The CSF1/CSF1R axis plays a role in this E to E/M transition, thus warranting testing its significance using an in vivo IBC model. Phenotypic transition and reversion between E and E/M phenotypes could be a new paradigm that reconciles two contradictory models of IBC metastasis. Citation Format: Kai K, Iwamoto T, Zhang D, Rao AUK, Thompson A, Sen S, Ueno NT. CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer [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 P4-14-02.

β‑carotene reverses tobacco smoke‑induced gastric EMT via Notch pathway in vivo.

Tobacco smoke is one of the serious risk factors of gastric cancer. Epithelial‑mesenchymal transition (EMT) has been shown to be associated with the initiation and carcinogenesis of gastric cancer. The role of Notch pathway in regulating tobacco smoke-induced EMT has not been investigated. β‑carotene, a carotenoid present in fruits, vegetables and rice, suppresses cancer progression. In this investigation, we evaluated the regulatory role of Notch pathway in tobacco smoke‑mediated gastric EMT and the preventive effect of β‑carotene using a BALB/c mouse smoking model. Exposure of mice to tobacco smoke reduced levels of epithelial markers, while the expression of mesenchymal markers were increased. We further found that Notch pathway modulated tobacco smoke-triggered EMT in the stomach of mice, as evidenced by these findings that tobacco smoke activated Notch activities, and tobacco smoke induced EMT was reversed by blocking Notch activities with FLI‑06. Moreover, treatment of β‑carotene prevented tobacco smoke‑mediated activation of Notch and EMT changes. Our data suggested that Notch regulate tobacco smoke induced gastric EMT and the protective effects of β‑carotene invivo. These findings may establish a new mechanism for tobacco smoke-associated gastric tumorigenesis and its chemoprevention.