Transcriptional Repressor Of E-cadherin Research Articles

Epidermal growth factor induces the initiation of epithelial mesenchymal transition in high-density colorectal cancer cell culture.

Epithelial-mesenchymal transition (EMT) is a step in the process through which colorectal cancer cells metastasize by gaining the cellular mobility associated with mesenchymal cells. However, whether the EMT occurs in cells tightly bound to each other remains largely unknown. In this study, we examined the dual influence of intercellular contact and epidermal growth factor (EGF) signaling on the induction of EMT in SW480 human colon carcinoma cells. Stimulation of densely cultured SW480 cells with EGF initiated partial EMT, following which E-cadherin levels were reduced. In these cells, the transcriptional repression of E-cadherin was caused by ZEB1 binding to its promoter region. EGF signaling did not directly induce ZEB1 mRNA upregulation but contributed to ZEB1 protein stability by regulating proteasomal degradation. Our findings indicate that EGF can induce EMT in colorectal cancer cells in the presence of cell-cell contact and may be a potential therapeutic target for metastasis.

Abstract 3704: The essential role of an alternate mRNA translation initiation in the regulation of breast cancer cell metabolism

Abstract Translational control and metabolic reprogramming are hallmarks of advanced cancers. Important genes involved in cancer progression express mRNAs that are selectively translated, including regulators of cancer cell metabolism. Cancer cells acquire an altered metabolism, switching from oxidative phosphorylation (OXPHOS) to a glycolytic phenotype (Warburg effect), to increase reliance on alternate metabolic pathways to support growth, proliferation, and metastasis. Triple-negative breast cancer (TNBC), one of the most aggressive and highly metastatic subtypes with the poorest outcome, is characterized by elevated glycolysis and low OXPHOS. Still, the exact mechanism for this metabolic switch is largely unknown. Using a TNBC cell model it has been shown an alternate mechanism of cap-dependent but mTORC1/eIF4Eindependent mRNA translation by the eIF4G homolog, DAP5, which directly binds the cap-binding protein eIF3d. Our research indicates that this alternate translation initiation is essential in regulating several mRNAs involved in breast cancer metabolism. DAVID analysis from genome-wide transcriptomic and translatomic studies, metabolomic assays including Glycolysis Assay [Extracellular Acidification], Oxidative Phosphorylation, and Mitotracker Assay, as well as RT-PCR and western blot analysis was performed from well-characterized triple-negative aggressive breast cancer cell lines,4T1 and MDA-231, Non-silencing (Nsi) control and silencing DAP5 (shDAP5) samples. Several mRNAs related to glucose metabolism decreased after silencing DAP5 and on the opposite, critical mRNAs associated with OXPHOS increased, independent of their steady-state mRNA abundance. Silencing DAP5 increased Oxidative Phosphorylation activity and decrease the glycolytic rate in TNBC breast cancer cells. Proteins correlated with aberrant glycolytic metabolism including the E-cadherin transcriptional repressors Snail (SNAI1) were reduced after silencing of DAP5. Our data indicates that the alternate mRNA translation initiation mechanism by DAP5 of specific mRNAs correlated to glycolysis and OXPHOS could play an essential role in the mechanism by which TNBC breast cancer cells orchestrate a metabolic switch required for malignant progression and metastasis. Citation Format: Hanna Rosenstock, Erna Mitaishvili, Columba de la Parra. The essential role of an alternate mRNA translation initiation in the regulation of breast cancer cell metabolism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3704.

CircPVT1 promotes silica-induced epithelial-mesenchymal transition by modulating the miR-497-5p/TCF3 axis.

Epithelial-mesenchymal transition (EMT) is a vital pathological feature of silica-induced pulmonary fibrosis. However, whether circRNA is involved in the process remains unclear. The present study aimed to investigate the role of circPVT1 in the silica-induced EMT and the underlying mechanisms. We found that an elevated expression of circPVT1 promoted EMT and enhanced the migratory capacity of silica-treated epithelial cells. The isolation of cytoplasmic and nuclear separation assay showed that circPVT1 was predominantly expressed in the cytoplasm. RNA immunoprecipitation assay and RNA pull-down experiment indicated that cytoplasmic-localized circPVT1 was capable of binding to miR-497-5p. Furthermore, we found that miR-497-5p attenuated the silica-induced EMT process by targeting transcription factor 3 (TCF3), an E-cadherin transcriptional repressor, in the silica-treated epithelial cells. Collectively, these results reveal a novel role of the circPVT1/miR-497-5p/TCF3 axis in the silica-induced EMT process in lung epithelial cells. Once validated, this finding may provide a potential theoretical basis for the development of interventions and treatments for pulmonary fibrosis.

Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action.

Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-β, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-β. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

Type 2 human papillomavirus E7 attenuates E-cadherin expression in human keratinocytes.

Human papillomaviruses (HPVs) are known to utilize the down-regulation of epithelial (E)-cadherin, a major component of adherens junctions of keratinocytes, to evade host immune surveillance in high-risk group. However, the effects of HPV on the function of E-cadherin in low-risk groups remain unknown. We investigated whether type 2 HPV (HPV-2) E7 could induce alterations in E-cadherin expression in transiently transfected keratinocytes and cell lines expressing HPV-2 E7. To examine the expression pattern of E-cadherin in cutaneous warts and normal skin samples, immunohistochemical analysis was performed. Quantitative real-time polymerase chain reactions, luciferase assays, western blot, immunocytochemistry, and electron microscopy were used to evaluate the mRNA and protein expression levels of E-cadherin in normal human epidermal keratinocytes transfected with HPV-2 E7 plasmid DNA or E7-specific siRNA and in E7-expressing cell lines. E-cadherin expression levels in HPV-2 positive cutaneous warts were significantly decreased compared to those in normal skin (p < 0.05). Similarly, the mRNA and protein expression levels of E-cadherin in E7 transiently transfected cells were significantly decreased compared to those in empty vector-transfected cells. The decreases were restored by transfection with E7-specific siRNA (p < 0.05). Likewise, cell lines expressing E7 showed a decreased expression of E-cadherin. When the cells were cultured in low attachment plates, cell-to-cell aggregation was inhibited. Taken together, our data suggest that HPV-2 E7, the causative agent of cutaneous warts, could mediate the transcriptional repression of E-cadherin.

Role of Prostatic Stem Cell Antigen (PSCA) and Snail in Different Prostatic Lesions) (An immunohistochemical study

Background: Prostatic carcinoma (PCa) represents the second most common cancer, and the fifth leading cause of cancer death among males worldwide. PSCA is a GPI-anchored cell surface protein. It belongs to the Thy-1/Ly-6 family which shows a functional diversity ranging from T-cell activation to apoptosis regulation. Snail is one of zinc finger proteins which are transcriptional repressors of E-cadherin. Aim: To study PSCA and Snail expression in different prostatic lesions to evaluate their roles in PCa. Material and Methods: This retrospective study was done upon 80 different prostatic lesions; 17 cases of BPH, 13 cases of HGPIN, and 50 cases of PCa. PSCA and Snail immunostaining was done and assessed for each case. Results: There was a highly significant statistical correlation between both PSCA and Snail expressions and histopathological type (P-value<0.01). PSCA expression showed a highly significant statistical correlation with Gleason score, tumor grade and stage (P-value<0.01), and a significant correlation with PSA, and peri-neural invasion (P-value<0.05). Snail expression showed a highly significant statistical correlation with Gleason score and tumor grade (P-value<0.01), and a significant correlation with lymph node metastasis and tumor stage (P-value<0.05). There was a highly significant statistical correlation between PSCA and Snail immune-expression (P-value<0.01). Conclusion: PSCA and Snail expressions correlate with the most important prognostic clinicopathological variables in PCa, thus they may represent a useful predictor of prognosis.

Cryo-EM structure of CtBP2 confirms tetrameric architecture

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly. Here, we present the cryoelectron microscopic structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is tetrameric. The physiological relevance of the observed tetramer was demonstrated in cell culture, showing that CtBP tetramer-destabilizing mutants are defective for cell migration, transcriptional repression of E-cadherin, and activation of TIAM1. Together with our cryoelectron microscopy studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

MARCKSL1 promotes the proliferation, migration and invasion of lung adenocarcinoma cells.

Lung cancer is the most common cancer in males and females and ~40% of lung cancer cases are adenocarcinomas. Previous studies have demonstrated that myristoylated alanine rich protein kinase C substrate (MARCKS) is upregulated in several types of cancer and is associated with poor prognosis in patients with breast cancer. However, its expression level and role in lung adenocarcinoma remain unknown. Therefore, the aim of the present study was to investigate the expression level and biological functions of MARCKS like 1 (MARCKSL1), a member of the MARCKS family, in lung adenocarcinoma. The expression level of MARCKSL1 was examined in human lung adenocarcinoma tissues and cell lines. MARCKSL1-specific small interfering RNAs effectively suppressed its expression level and significantly inhibited the proliferation, migration and invasion of lung adenocarcinoma cells. Additionally, the role of MARCKSLI in the regulation of metastasis was examined. Silencing MARCKSL1 decreased the expression of the epithelial-mesenchymal transition (EMT)-associated proteins E-cadherin, N-cadherin, vimentin and snail family transcriptional repressor 2, and decreased the phosphorylation level of AKT. The results obtained in the current study suggested that MARCKSL1 promoted the progression of lung adenocarcinoma by regulating EMT. MARCKSLI may have prognostic value and serve as a novel therapeutic target in lung adenocarcinoma.

Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion.

Previously, it has been reported that homeobox A9 (HOXA9) protein expression is downregulated in lung cancer cells, and that its expression is inversely correlated with the metastatic potential of lung cancer cells both in vitro and in vivo. As such, HOXA9 shows therapeutic potential. The development of therapeutic strategies based on this protein is, however, limited due to its poor membrane permeability. To overcome this problem, we developed a system to deliver HOXA9 protein into non-small cell lung cancer (NSCLC) cells. First, we constructed a delivery vector expressing polyarginine, a cell-penetrating peptide, as well as HOXA9. The resulting recombinant R10-HOXA9 protein was effectively introduced into A549 and NCI-H1299 NSCLC cells. Next, we examined the roles and molecular mechanisms of recombinant R10-HOXA9 in processes involved in tumor progression. To investigate the therapeutic efficacy of the delivery system, we performed cell motility assays using both in vitro and in vivo experimental models. We found that recombinant R10-HOXA9 protein reduced the invasion and migration rate, but not the proliferation rate, of the NSCLC cells tested, both in vitro and in vivo. Treatment of NSCLC cells with recombinant R10-HOXA9 protein led to a significant increase in E-cadherin expression. Conversely, we found that the expression of snail family zinc finger 2 (SLUG), a transcriptional repressor of E-cadherin, was markedly decreased. In an experimental metastatic mouse model, recombinant R10-HOXA9 protein was found to effectively reduce the rate of lung cancer cell motility. Our data suggest that the developed cell-permeable R10-HOXA9 system may serve as a useful tool to prevent NSCLC cell migration and invasion.

The metastasis suppressor, NDRG1, attenuates oncogenic TGF-β and NF-κB signaling to enhance membrane E-cadherin expression in pancreatic cancer cells.

The metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-β (TGF-β) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and β-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and β-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/β and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-β signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential.

Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer

Colorectal cancer (CRC) is the most common malignant disease worldwide due to its metastasis via the epithelial-mesenchymal transition (EMT) process. E-cadherin and Wnt signaling are emerging as potential targets for suppressing the EMT. In this context, Axin2 has been recognized as a negative regulator that inhibits glycogen synthase kinase 3β (GSK3β)-mediated degradation of Snail1, a transcriptional repressor of E-cadherin. However, Axin2 can also impede Wnt signaling via β-catenin degradation. Therefore, Axin2 may serve as either a promoter or suppressor of tumors, and the effects of its inhibition on the cell proliferation and metastasis of CRC require further elucidation. Here, esculetin (ES), a coumarin, was found to have the most potential effects on both β-catenin-responsive transcriptional and E-cadherin promoter activities. ES also showed anti-proliferative and anti-invasive activities in CRC cells. Mechanistically, Axin2 suppression by ES contributed to E-cadherin-mediated Wnt signaling inhibition. Moreover, the ability of ES to inhibit tumor growth and metastasis via Axin2 suppression was further supported in an HCT116-implanted orthotopic mouse model. Collectively, these findings suggest that targeting the Axin2/E-cadherin axis by ES may be an attractive therapeutic strategy for the treatment of metastatic CRC.

CD44 exerts a functional role during EMT induction in cisplatin-resistant head and neck cancer cells.

A number of studies report that epithelial to mesenchymal transition (EMT) supports the generation and maintenance of cancer stem cells (CSCs), which show tumor seeding ability and drug resistance; however, the molecular mechanisms underlying induction of EMT-associated tumor malignancy remain unclear. The present study reports that oral cancer cells switch from expressing the CD44 variant form (CD44v) to expressing the standard form (CD44s) during acquisition of cisplatin-resistance, which resulted in EMT induction. CD44s induced an EMT phenotype in cisplatin resistant cells by up-regulating ZEB1, a transcriptional repressor of E-cadherin. More importantly, CD44s up-regulated ZEB1 by suppressing microRNA-200c, which is a non-coding RNA that directly represses the ZEB1 gene. These results demonstrate the importance of the association between platinum resistance and CD44s during EMT induction in oral cancer cells.

Hyperuricemia and Progression of Chronic Kidney Disease: Role of Phenotype Transition of Renal Tubular and Endothelial Cells.

Although the clinical implication of hyperuricemia in chronic kidney disease has been an issue of active debate, recent data suggested a causative role of uric acid (UA) in the development of renal disease. Afferent arteriopathy, an induction of oxidative stress and an activation of local inflammation, have been regarded as the mechanisms of UA-induced renal disease, which contribute to glomerular hypertrophy and interstitial fibrosis via endothelial dysfunction. However, there have been rare studies on the direct effect of UA on phenotype transition of renal cells such as epithelial-to-mesenchymal transition (EMT) or endothelial-to-mesenchymal transition (EndoMT). We have reported that UA-induced EMT of cultured renal tubular cells, which was blocked by the organic anion transport inhibitor, probenecid. UA increased the expression of snail and slug, the transcriptional repressors of E-cadherin, which resulted in a downregulation of E-cadherin production. UA also increased the degradation of E-cadherin via ubiquitination. UA also induced EndoMT with an increase in ROS generation and glycocalyx shedding of cultured vascular endothelial cells. Treatment with antioxidants ameliorated UA-induced EndoMT. In the kidney of hyperuricemic rats, there was an evidence of EMT before the development of significant tubulointerstitial fibrosis, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA) in renal tubular cells. Allopurinol significantly inhibited UA-induced EMT with an amelioration of renal fibrosis. In addition, endothelial staining in peritubular capillaries (PTC) was substantially decreased with de-novo expression of α-SMA in endothelial cells of PTC. Key Messages: UA per se induced a phenotypic transition of epithelial and endothelial cells via an induction of oxidative stress and glycocalyx shedding, which could be one of the mechanisms of UA-induced kidney disease.

MiR-2392 suppresses metastasis and epithelial-mesenchymal transition by targeting MAML3 and WHSC1 in gastric cancer.

MicroRNAs have emerged as essential regulators of various cellular processes. We identified the role and underlying mechanisms of miR-2392 in gastric cancer (GC) metastasis. MiR-2392 was down-regulated in GC cell lines and tissues, and overexpression of miR-2392 significantly inhibited GC invasion and metastasis in vitro and in vivo We identified MAML3 and WHSC1 as novel targets of miR-2392, and knockdown of MAML3 and WHSC1 had the same antimetastatic effect as that of miR-2392 in GC cells. These effects were clinically relevant, as low miR-2392 expression was correlated with high MAML3 and WHSC1 expression and poor survival in patients with GC. Furthermore, forced expression of miR-2392 substantially suppressed Slug and Twist1, transcriptional repressors of E-cadherin, by targeting MAML3 and WHSC1, respectively, resulting in inhibition of the epithelial-mesenchymal transition. These findings indicate that the miR-2392-MAML3/WHSC1-Slug/Twist1 regulatory axis plays a critical role in GC metastasis. Restoration of miR-2392 may be a therapeutic approach for blocking GC metastasis.-Li, J., Li, T., Lu, Y., Shen, G., Guo, H., Wu, J., Lei, C., Du, F., Zhou, F., Zhao, X., Nie, Y., Fan, D. MiR-2392 suppresses metastasis and epithelial-mesenchymal transition by targeting MAML3 and WHSC1 in gastric cancer.

Abstract 3336: Comparison of epithelial mesenchymal transition mediated TKI resistanc NSCLC cells containing wild type EGFR and mutant EGFR

Abstract Epithelial to mesenchymal transition (EMT) is a vital process in development and of metastasis and occurs when epithelial cell lose their polarized structure, by reducing adherent junction proteins E-cadherin, Claudin and ZO-1 on the membrane. Cells with EMT are elongated spindle like structures due to upregulation of mesenchymal markers Vimentin and N-cadherin. EMT may be responsible for resistance to molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) against EGFR which is used in patients with activated EGFR mutations. However, these patients acquire resistance to TKIs after prolonged use. This acquired resistance to TKIs may also be due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. EMT is regulated by p120-catenin which interacts with the Kaiso factor in the cell nucleus and inhibits the transcriptional repressor activity of Kaiso factor. Kaiso factor represses Wnt target genes such as ZEB-1. The binding of p120-catenin to Kaiso factor also increases Wnt signaling which results in loss of E-cadherin. Cells undergoing EMT can acquire cancer stem-cell like characteristics by expressing stem-cell marker ABCB1. Thus we investigated EMT characteristics in TKI-resistant NSCLC cells, H2170 ER (Erlotinib resistant), H358 ER and H1975. To determine modulation of EMT biomarkers in TKI-resistant cells, H1975 with L858R and T790M mutations was compared to TKI-sensitive cell line H3255 with L858R mutation, using immunoblotting, and qPCR. Expression of stem-cell markers ABCB1 and EMT biomarker E-cadherin are measured using flow cytometry. Key EMT-related proteins such as PRMT-1, Slug, Snail, Twist, p120-catenin, and Vimentin were upregulated by 3.2, 3.18, 6.2, 1.68, 4 and 6.5 fold, respectively, and E-cadherin, Claudin and ZO-1 were downregulated by 89%, 90% and 99% fold as compared to the H3255 TKI-sensitive cell line. We also observed upregulation of N-cadherin, ZEB-1 and Vimentin by 2.4, 2.6, and 12 fold and downregulation of E-cadherin by 50% in H1975 by qPCR. Immunofluorescence studies for Vimentin showed that H1975 cells were more elongated and stratified as compared to the H3255 that had polarized structures. 90% colocalization of p120-catenin and Kaiso factor was seen H1975 cells whereas 10% colocalization was seen in H3255 cells. Flow cytometry results indicated that there was significant increase in expression of stem cell marker ABCB1 in TKI resistant cells H2170 ER, H358 ER and H1975 in comparison to H2170 P(Parental), H358 P and H3255 TKI sensitive cells. In conclusion, our results indicate that EMT is mediated through PRMT-1, which methylates Twist, transcriptional repressor of E-cadherin along with Slug and Snail in H1975 cells with T790M mutation. Cancer stem-cell marker ABCB1 is specific for TKI resistant NSCLC cells which exhibit EMT. Citation Format: Tsatsral Iderzorig, Sanjana Singh, Gagan Chhabra, Neelu Puri. Comparison of epithelial mesenchymal transition mediated TKI resistanc NSCLC cells containing wild type EGFR and mutant EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2017-3336

Sprouty2 inhibits amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells.

Similar to Drosophila Sprouty (SPRY), mammalian SPRY proteins inhibit the receptor tyrosine kinase-mediated activation of cellular signaling pathways. SPRY2 expression levels have been shown to be down-regulated in human ovarian cancer, and patients with low SPRY2 expression have significantly poorer survival than those with high SPRY2 expression. In addition, epidermal growth factor receptor (EGFR) is overexpressed in human ovarian cancer and is associated with more aggressive clinical behavior and a poor prognosis. Amphiregulin (AREG), the most abundant EGFR ligand in ovarian cancer, binds exclusively to EGFR and stimulates ovarian cancer cell invasion by down-regulating E-cadherin expression. However, thus far, the roles of SPRY2 in AREG-regulated E-cadherin expression and cell invasion remain unclear. In the present study, we show that treatment with AREG up-regulated SPRY2 expression by activating the EGFR-mediated ERK1/2 signaling pathway in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, overexpression of SPRY2 attenuated the AREG-induced down-regulation of E-cadherin by inhibiting the induction of the E-cadherin transcriptional repressor, Snail. Moreover, SPRY2 overexpression attenuated AREG-stimulated cell invasion and proliferation. This study reveals that SPRY2 acts as a tumor suppressor in human ovarian cancer and illustrates the underlying mechanisms that can be used as possible targets for the development of novel therapeutics.

MED28 Regulates Epithelial-Mesenchymal Transition Through NFκB in Human Breast Cancer Cells.

MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium-derived solid tumor, migration and invasion are preceded by the progression of epithelial-mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast-like MDA-MB-231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E-cadherin. The suppression effect was also accompanied by downregulation of p-NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme-like appearance correlated with increased expression of MED28, p-p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E-cadherin and MMP9 in MCF7 cells. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. J. Cell. Physiol. 232: 1337-1345, 2017. © 2016 Wiley Periodicals, Inc.

Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells.

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits.Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/β-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1.Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.

Growth differentiation factor 8 induces SKOV3 ovarian cancer cell migration and E-cadherin down-regulation

Epithelial ovarian cancer is the most lethal gynecological malignancy because most women present with late stage disseminated disease. Epithelial-mesenchymal transition (EMT) is characterized by the down-regulation of E-cadherin and up-regulation of N-cadherin, and is a crucial event in the pathogenesis of ovarian cancer. Transforming growth factor-β (TGF-β) is a major regulator of EMT in many normal and neoplastic cell types. Growth differentiation factor 8 (GDF8), which also activates TGF-β-like SMAD2/3 signaling, is best known for negatively regulating muscle growth. Though recent studies suggest that GDF8 enhances placental trophoblast cell migration, little is known about the role of GDF8 in EMT and cancer metastasis. We hypothesized that GDF8 could enhance ovarian cancer cell migration by inducing EMT. Here we demonstrate for the first time that GDF8 down-regulates E-cadherin but does not alter N-cadherin in SKOV3 ovarian cancer cells. This effect is abolished by the activin receptor-like kinase (ALK)4/5/7 inhibitor SB431542 or siRNA-mediated knockdown of ALK5, whereas knockdown of ALK4 is only partially inhibitory. GDF8 treatment increases the phosphorylation of SMAD2/3 and up-regulates the E-cadherin transcriptional repressors Snail and Slug; and these effects are abolished by pre-treatment with SB431542. Knockdown of common SMAD4 fully reverses the effects of GDF8 on E-cadherin and partially attenuates its effects on Snail and Slug. Importantly, GDF8 treatment increases SKOV3 cell migration and this effect is blocked by SB431542. Our study suggests that GDF8 promotes ovarian cancer cell migration via ALK4/5-SMAD2/3-E-cadherin signaling.

PIPKIγ and talin couple phosphoinositide and adhesion signaling to control the epithelial to mesenchymal transition.

Epithelial cells acquire migratory/invasive and stemness traits upon conversion to the mesenchymal phenotype. The expression of E-cadherin is a key to this transition; yet precise understanding of the pathways involved in integrating E-cadherin loss to the gain of mesenchymal traits remains poorly understood. Here, we show that phosphoinositide-generating enzyme, PIPKIγ, expression is upregulated upon epithelial-mesenchymal transition (EMT) and together with the cytoskeletal protein talin assemble into a signaling complex upon E-cadherin loss. PIPKIγ and talin together control the adhesion and phosphoinositide signaling that regulates conversion to the mesenchymal phenotypes. PIPKIγ and talin regulate the stability of E-cadherin transcriptional repressors, snail and slug, induced by transforming growth factor-β1 or extracellular matrix protein. Loss of PIPKIγ or talin or their interaction impaired EMT and the acquisition of cell motility and stemness. This demonstrates a mechanism where a phosphoinositide-generating enzyme PIPKIγ couples with a cytoskeletal protein talin to control the acquisition of mesenchymal phenotypes.