Loss Of E-cadherin Research Articles

Detailed analyses about predictive value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography in invasive lobular breast cancer

Backgrounds: Management of invasive lobular breast cancer (ILC) is limited with only using standard uptake value (SUV) max of 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography. We aimed detailed predictive, prognostic and clinical value of parameters, metabolic tumor volume (MTV), and total lesion glycolysis (TLG) with survival data. Methods: Forty-nine ILC was included in this study. SUVmax, MTV, and TLG documented in molecular and histopathological subtypes. SUVmax, MTV, and TLG compared with nodal involvement, distant metastasis, estrogen receptor (ER), progesterone receptors (PR) and HER status, Ki-67 and survival data. Results: 61.2% luminal A, 36.7% luminal B and 2.0% TN. 53.1% classic and 46.9% pleomorphic. 93.9% ER + and 81.6% had e-cadherin loss. Mean SUVmax was 8.32 ± 4.73. No statistically significant relationship found between classical and pleomorphic with SUVmax (P = 0.616). FDG uptake found significantly higher with tumor >2 cm (P = 0.039). Luminal B SUVmax significantly higher than luminal A (P = 0.013). PR-, Ki-67 high expression, axillary involvement and luminal B significantly reduced survival time (P = 0.034, 0.019, 0.032, and 0.005, respectively). Statistically significant correlation found between high MTV and TLG, tumor diameter (TD) (P = 0.001 and 0.000, respectively). Conclusions: Loss e-cadherin and pleomorphic types did not affect the prognosis. ILC shows low FDG sensitivity compared other breast cancer types, it found to be significantly associated with prognostic factors; TD, molecular subtype, Ki-67 and metastasis. Although volumetric parameters did not add extrapredictive value to FDG involvement pattern, it was found that method remains in diagnosis, staging, and treatment follow-up of ILC.

Comparison of E-cadherin and CD56 Expression in Papillary Thyroid Carcinoma and Non-Neoplastic Thyroid Lesions

Introduction: Papillary Thyroid Carcinomas (PTC) are the most common among thyroid malignancies. The incidence of this tumour is rapidly increasing around the world. Kerala has the highest incidence of this tumour in India. Abnormalities in adhesion molecules, E-cadherin and CD56 have recently been implicated in thyroid tumourigenesis. Sometimes the diagnosis of PTC is difficult, as there are a good number of histological variants and some may be encapsulated. In such situations, evaluation of the expression of adhesion molecules like E-cadherin and CD56 are useful in accurate diagnosis. Aim: To study the Immunohistochemical Expression of E-cadherin and CD56 in PTC, its adjacent normal thyroid tissue and other non-neoplastic thyroid lesions. Materials and Methods: This was a descriptive study conducted at Amala Institute of Medical Sciences, Thrissur from January 2018 to June 2019. Seventy six thyroidectomy specimens (38 each of PTC and Non-neoplastic lesions) were studied after satisfying the inclusion and exclusion criteria. Microscopic examination of the Haematoxylin and Eosin stained sections were done for selecting the representative tissue block to immunostain for E-cadherin and CD56. Statistical analysis was performed using Chi-square test and Fisher’s-exact tests. P-value of <0.05 was considered as significant. Results: E-cadherin expression was negative in 37 cases of PTC. Only two non-neoplastic lesions were negative for E-cadherin (p-value of 0.021). No significant correlation was observed between E-cadherin expression and poor prognostic factors (tumour diameter, multifocality, extrathyroidal extension and lymph node metastasis). All PTC cases showed negative CD56 expression. A total of 34 out of 38 Non-neoplastic cases showed positive CD56 staining (significant p value=0.011). In non-neoplastic lesions, E-cadherin and CD56 were preserved. CD56 showed highest specificity (100%). Conclusion: PTC was characterised by decreased or absent expression of E-cadherin as compared to the adjacent thyroid tissue. CD56 expression was uniformly negative in all the PTC cases. CD56 marker had highest specificity (100%). In follicular patterned thyroid lesions, CD56 as a single marker may be useful for identifying PTC from other thyroid lesions in daily practice. To conclude, CD56 negativity and E-cadherin loss can assist in decision making of difficult cases.

Влияние синдрома обструктивного апноэ/гипопноэ сна на экспрессию Е-кадгерина в эпителии пищевода

Aim: to evaluate the effect of obstructive sleep apnea-hypopnea syndrome (OSAHS) on E-cadherin expression and epithelium histoarchitectonics of the lower third of the esophagus. Patients and Methods: 108 patients were included in a cross-sectional comparative study. According to the results of somnology, a mild severity of OSAHS was diagnosed in 49 patients (group 1), an average severity — in 29 (group 2). Group 3 included 30 people without OSAHS. All patients underwent esophagogastroduodenoscopy with a biopsy of the lower third of the esophagus. Morphometrics of epithelial thickness, height of connective tissue papillae, and internuclear distances in all epithelial layers was performed. To assess the E-cadherin expression, immunohistochemistry was performed using monoclonal antibodies to E-cadherin. Results: in contrast to the comparison group, patients with mild and moderate OSAHS were characterized by a higher epithelium thickness (273 (228; 372), 360 (312; 411), 186 (168; 192) μm, respectively, р1–3=0.000, р2–3=0.000), higher connective tissue papillae (108 (96; 120), 144 (120; 228), 72 (60; 84) μm, respectively, р1–3=0,001, р2–3=0,000), and larger internuclear distances in superficial layer (9 (7; 10), 8 (7; 9), 6 (5; 7) μm, respectively, р1–3=0.000, р2–3=0.002), spinous layer (8 (6; 9), 8 (7; 8), 6 (5; 7) μm, respectively, р1–3=0.000, р2–3=0.000), basal layer (4 (3; 5), 3 (3; 4), 2 (2; 3) μm, respectively, р1–3=0.000, р2–3=0,001) of epithelium. E-cadherin expression decreased in patients with both mild OSAHS (0.748 (0.623; 0.833) and 0.539 (0.263; 0.676), respectively, p=0.044) and moderate OSAHS (0.748 (0.623; 0.833) and 0.163 (0.026; 0.365), respectively, p=0.000) versus the comparison group. Correlations were found between respiratory monitoring indicators and E-cadherin expression in the esophageal epithelium. Conclusion: OSAHS negatively affects the lining condition of the lower third of the esophagus, contributes to the loss of E-cadherin, which creates conditions for disorganization of epithelial tissue and increases the risk of precancerous diseases and esophageal cancer. KEYWORDS: obstructive sleep apnea-hypopnea syndrome, E-cadherin, esophageal epithelium, histoarchitectonics, epithelial-mesenchymal transition, intermittent hypoxia, apnea-hypopnea index. FOR CITATION: Shelkovich Yu.Ya. Effect of obstructive sleep apnea-hypopnea syndrome on E-cadherin expression in esophageal epithelium. Russian Medical Inquiry. 2020;4(5):268–273. DOI: 10.32364/2587-6821-2020-4-5-268-273.

RAS-mediated suppression of PAR3 and its effects on SCC initiation and tissue architecture occur independently of hyperplasia.

Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Invasive Lobular Breast Cancer as a Distinct Disease: Implications for Therapeutic Strategy.

Invasive lobular carcinoma comprises 10–15% of all breast cancers and is increasingly recognised as a distinct and understudied disease compared with the predominant histological subtype, invasive ductal carcinoma. Hallmarks of invasive lobular carcinoma include E-cadherin loss, leading to discohesive morphology with cells proliferating in single-file strands and oestrogen receptor positivity, with favourable response to endocrine therapy. This review summarises the distinct histological and molecular features of invasive lobular carcinoma with focus on diagnostic challenges and the impact on surgical management and medical therapy. Emphasis is placed on recent advances in our understanding of the unique molecular biology of lobular breast cancer and how this is optimising our therapy approach in the clinic.

Epithelial-Mesenchymal Transition in Skin Cancers: A Review.

Epithelial-mesenchymal transition (EMT) is involved in physiologic processes such as embryogenesis and wound healing. A similar mechanism occurs in some tumors where cells leave the epithelial layer and gain mesenchymal particularities in order to easily migrate to other tissues. This process can explain the invasiveness and aggressiveness of these tumors which metastasize, by losing the epithelial phenotype (loss of E-cadherin, desmoplakin, and laminin-1) and acquiring mesenchymal markers (N-cadherin). Complex changes and interactions happen between the tumor cells and the microenvironment involving different pathways, transcription factors, altered expression of adhesion molecules, reorganization of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in specific microRNAs. The purpose of this review is to determine particularities of the EMT process in the most common malignant cutaneous tumors (squamous cell carcinoma, basal cell carcinoma, and melanoma) which still have an increasingly high incidence. More studies are required on this topic in order to establish clear correlations. High costs related to skin cancer therapies in general as well as high impact on patients' quality of life demand finding new, reliable prognostic and therapeutic markers with significant public health impact.

Cadmium and Lead Decrease Cell-Cell Aggregation and Increase Migration and Invasion in Renca Mouse Renal Cell Carcinoma Cells.

Metastatic renal cell carcinoma (RCC) remains an important clinical issue; the 5-year survival rate of patients with metastasis is approximately 12%, while it is 93% in those with localized disease. There is evidence that blood cadmium and lead levels are elevated in RCC. The current studies were designed to assess the impact of cadmium and lead on the progression of RCC. The disruption of homotypic cell–cell adhesion is an essential step in epithelial-to-mesenchymal transition and tumor metastasis. Therefore, we examined the impact of cadmium and lead on the cadherin/catenin complex in Renca cells—a mouse RCC cell line. Lead, but not cadmium, induced a concentration-dependent loss of E-cadherin, while cadmium, but not lead, increased p120-catenin expression, specifically isoform 1 expression. Lead also induced a substantial increase in matrix metalloproteinase-9 levels. Both cadmium and lead significantly decreased the number of Renca cell aggregates, consistent with the disruption of the cadherin/catenin complex. Both metals enhanced wound healing in a scratch assay, and increased cell migration and invasion. These data suggest that cadmium and lead promote RCC progression.

Abstract C34: Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit hypoxia due to high oxygen demand and oxygen diffusional limitations (1). Hypoxia has been reported as a driver of epithelial-mesenchymal transition (EMT) (2), which occurs early in pancreas cancer dissemination and promotes resistance to chemotherapy (3,4). Druggable signaling pathways that promote EMT in the hypoxic PDAC microenvironment may therefore represent useful targets for combination therapy. Our lab previously studied the roles of ERK and SHP2 in driving EMT in response to growth factors (5,6). More recently, our lab employed an unbiased data-driven modeling approach and found that the ERK, JNK, and NF-kappaB pathways coordinate to drive robust EMT in PDAC cells treated with combinations of growth factors (unpublished). In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to a shift from normoxic to hypoxic conditions. The immortalized human PDAC cell line HPAF-II, which exhibits baseline epithelial characteristics, was used for these studies. In simple two-dimensional cell culture, a shift from 20% to 1% oxygen for ≤120 hr led to several indications of EMT, including decreased epithelial cell cluster circularity, reduced junctional E-cadherin expression and an increased fraction of vimentin-positive cells (determined by an automated image analysis pipeline), and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2 (determined by RT-qPCR). A hypoxic cellular response was confirmed by immunoblotting, which showed an early (4 hr) peak in HIF-1alpha expression and a relatively late (48 hr) peak in HIF-2alpha expression, as well as increased expression of the HIF target genes PGK1 and SLC2A1. In three-dimensional cultures of HPAF-II cell spheroids in a collagen I matrix, a shift to hypoxia promoted cell invasion into the matrix, a behavior also observed when HPAF-II cells were treated with exogenous growth factors that drive EMT. To probe for the activation of the ERK, JNK, and NF-kappaB pathways, we used a combination of immunoblotting and immunofluorescence microscopy. A shift from 20% to 1% oxygen increased the phosphorylation of ERK and promoted the nuclear localization of c-Jun, a substrate of JNK. Inhibition of MEK or JNK at the same time as the shift to 1% oxygen prevented the loss of junctional E-cadherin that otherwise occurred, with JNK inhibition having the greater effect. JNK inhibition also abrogated loss of CDH1 transcripts and the increase of VIM transcripts otherwise observed in response to hypoxia. Thus, activation of the ERK and JNK pathways in PDAC cells in response to a shift to low oxygen conditions promoted EMT. These pathways may therefore represent useful targets for antagonizing EMT in the hypoxic microenvironment characteristic of PDAC tumors.

Abstract C22: Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway

Abstract Metastasis is one of the main reasons for poor survival in patients with pancreatic ductal adenocarcinoma (PDA). However, the molecular mechanism of PDA metastasis remains elusive. Recent studies failed to find metastasis-specific genetic mutations, and thus suggested that aberrant epigenetic regulation may be one of the crucial mechanisms. Many epigenetic regulator genes are altered in PDA, including KDM6A, a histone H3K27 demethylase, which has been identified by whole-genomic mutational analyses as a driver of PDA. Recent studies have shown that loss of KDM6A induces aggressive squamous-like PDA by activing superenhancers and promotes metastasis. However, the underlying mechanism by which KDM6A promotes metastasis remains elusive. Using stable KDM6A knockout PDA cell lines established by CRISPR/Cas9 system and siRNA, we knocked out or knocked down the expression of KDM6A in PDA cells. We also established an inducible KDM6A re-expressed PDA cell line that has homozygous KDM6A deletion. A novel Bru-seq technology was used to determine global changes of nascent RNA synthesis in KDM6A-null cells. Cell migration and invasion was assessed in vitro. To study the impact of KDM6A loss in vivo, we crossed KDM6Afl/fl mice with the well-established Ptf1aCre; Kras G12D; p53 R172H (KPC) PDA mouse model. Bru-seq data have shown that epithelial-mesenchymal transition (EMT) pathway is highly upregulated in KDM6A knockout cells and downregulated in KDM6A re-expressed PDA cells. EMT is a well-known process that is involved in tumor metastasis. KDM6A knockout cells demonstrated decreased cell proliferation but increased cell migration and invasion with mesenchymal cell morphology changes in vitro. Hallmark epithelial marker E-cadherin was significantly downregulated in KDM6A knockout cells, supporting the EMT process. Furthermore, genetic mouse model showed that mice that lose KDM6A expression developed PDA and metastasis much earlier than KPC mice. The morphology of the PDA developed in KDM6A-null KPC mice is more sarcomatoid or mesenchymal compared to PDA in KPC mice. Immunohistochemistry analysis also demonstrated loss of epithelial marker E-cadherin and gain of mesenchymal marker vimentin in tumor cells. In summary, these results suggest that loss of KDM6A promotes PDA development and metastasis by upregulating EMT signaling. These data linked epigenetic regulation to EMT signaling and provided insights into the role of KDM6A in PDA development. These novel findings also provided foundation for developing new therapeutic strategies. Citation Format: Sivakumar Jeyarajan, Zhujun Yi, Shanqiao Wei, Shuang Lu, Hong Kim, Michelle Paulsen, Karan Bedi, Mats Ljungman, Jiaqi Shi. Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C22.

Molecular Predictors for Advanced Papillary Thyroid Carcinoma Recurrence.

Despite its indolent course, one-third of the papillary thyroid carcinoma (PTC) cases relapses, which directly impact on the quality of patients' lives. The molecular predictors of recurrence of PTC are poorly defined. We aimed at evaluating the long-term (10–20 years) prognostic value of aggressiveness markers in advanced PTC. To this end, immunohistochemistry for BRAFV600E, Estrogen receptor α, Progesterone receptor, Ki-67, and E-cadherin were performed in 53 primary advanced PTC from an up to 20 years follow-up patients from a well-characterized Brazilian cohort. Categorical data were summarized using frequencies and groups were compared using Chi-squared and Fisher's exact tests. The expressions of the aggressiveness markers were associated with clinical-pathological data using the single-covariate logistic regression analysis. The Kaplan-Meier method with the Log-rank and Peto tests was used to estimate the probability of PTC-free survival. Persistence and recurrence (active disease) were associated with age (≥55 years), tumor size (>2 cm), extrathyroidal extension, local aggressiveness, macroscopic lymph node metastasis, and TNM stage at initial treatment. The BRAFV600E mutation status was associated with extrathyroidal extension, local aggressiveness, and inversely associated with distant metastasis at initial treatment. All progesterone receptor-positive patients had active disease and displayed a shorter time of PTC-free survival than the negative ones using the Kaplan-Meir analysis (p = 0.001, Log Rank; p = 0.005, Peto). Loss of E-cadherin expression was associated with an increase in the probability of active disease (OR = 3.75). BRAFV600E could be useful as a biomarker of local aggressiveness, while PR positive and E-cadherin loss of expression could predict the recurrence of advanced PTC.

\u03b2-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

WNT signaling activation in colorectal cancers (CRCs) occurs through APC inactivation or β-catenin mutations. Both processes promote β-catenin nuclear accumulation, which up-regulates epithelial-to-mesenchymal transition (EMT). We investigated β-catenin localization, transcriptome, and phenotypic differences of HCT116 cells containing a wild-type (HCT116-WT) or mutant β-catenin allele (HCT116-MT), or parental cells with both WT and mutant alleles (HCT116-P). We then analyzed β-catenin expression and associated phenotypes in CRC tissues. Wild-type β-catenin showed membranous localization, whereas mutant showed nuclear localization; both nuclear and non-nuclear localization were observed in HCT116-P. Microarray analysis revealed down-regulation of Claudin-7 and E-cadherin in HCT116-MT vs. HCT116-WT. Claudin-7 was also down-regulated in HCT116-P vs. HCT116-WT without E-cadherin dysregulation. We found that ZEB1 is a critical EMT factor for mutant β-catenin-mediated loss of E-cadherin and Claudin-7 in HCT116-P and HCT116-MT cells. We also demonstrated that E-cadherin binds to both WT and mutant β-catenin, and loss of E-cadherin releases β-catenin from the cell membrane and leads to its degradation. Alteration of Claudin-7, as well as both Claudin-7 and E-cadherin respectively caused tight junction (TJ) impairment in HCT116-P, and dual loss of TJs and adherens junctions (AJs) in HCT116-MT. TJ loss increased cell motility, and subsequent AJ loss further up-regulated that. Immunohistochemistry analysis of 101 CRCs revealed high (14.9%), low (52.5%), and undetectable (32.6%) β-catenin nuclear expression, and high β-catenin nuclear expression was significantly correlated with overall survival of CRC patients (P = 0.009). Our findings suggest that β-catenin activation induces EMT progression by modifying cell-cell junctions, and thereby contributes to CRC aggressiveness.

Characterization and printability of Sodium alginate -Gelatin hydrogel for bioprinting NSCLC co-culture

3D bioprinting improves orientation of in vitro tumor models by offering layer by layer positioning of cancer cells and cancer associated fibroblasts (CAFs) which can replicate tumor microenvironment. Aim of this study was to develop a sodium alginate -gelatin (SA-GL) hydrogel by optimizing rheological parameters to print non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung CAFs co-cultures. SA-GL hydrogels were prepared, and rheological properties were evaluated. Both the cells were mixed with the hydrogel and printed using INKREDIBLE bioprinter. Hydrogels prepared with 3.25% and 3.5% (w/v) SA and 4% (w/v) GL showed higher printability and cell viability. A significant decline in viscosity with shear rate was observed in these hydrogels suggesting the shear thinning property of hydrogels. Spheroid size distribution after 15 days was in the diameter range of 50–1100 µm. Up-regulation of vimentin, α-SMA and loss of E-cadherin in co-culture spheroids confirmed cellular crosstalk. This study demonstrates that rheological optimization of SA-GL hydrogel enhances printability and viability of NSCLC PDX and CAF co-culture which allows 3D co-culture spheroid formation within the printed scaffold. Therefore, this model can be used for studying high throughput drug screening and other pre-clinical applications.

Expression of E-cadherin and specific CXCR3 isoforms impact each other in prostate cancer

BackgroundCarcinoma cells shift between epithelial and mesenchymal phenotypes during cancer progression, as defined by surface presentation of the cell-cell cohesion molecule E-cadherin, affecting dissemination, progression and therapy responsiveness. Concomitant with the loss of E-cadherin during the mesenchymal transition, the predominant receptor isoform for ELR-negative CXC ligands shifts from CXCR3-B to CXCR3-A which turns this classical G-protein coupled receptor from an inhibitor to an activator of cell migration, thus promoting tumor cell invasiveness. We proposed that CXCR3 was not just a coordinately changed receptor but actually a regulator of the cell phenotype.MethodsImmunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.ResultsWe found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients’ tissue.ConclusionsCXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor’s phenotype.

Engagement of Robo1 by Slit2 induces formation of a trimeric complex consisting of Src-Robo1-E-cadherin for E-cadherin phosphorylation and epithelial-mesenchymal transition

Loss of E-cadherin elicits epithelial-mesenchymal transition (EMT). While both the Src family of membrane-associated non-receptor tyrosine kinases (SFKs) and Slit2 binding to Roundabout 1 (Robo1) have been shown to induce E-cadherin repression and EMT, whether these two signaling pathways are mechanistically coupled remains unknown in epithelial cells. Here we found that Slit2 and Robo1 overexpression activated Src kinases for tyrosine phosphorylation, degradation of E-cadherin and induction of EMT. Specific blockade of Slit2 binding to Robo1 inactivated Src, prevented E-cadherin phosphorylation and EMT induction. Biochemically, the cytoplasmic CC3 motif of Robo1 (CC3) bound directly to the SH2 and 3 domains of c-Src and the cytoplasmic domains of E-cadherin. Slit2 induced Robo1 association with endogenous c-Src and E-cadherin, whereas ectopic expression of CC3 dissociated this protein complex in colorectal epithelial cells. These results indicate that Slit2 not only induces Robo1 binding to Src, but also recruits Src to E-cadherin for tyrosine phosphorylation of E-cadherin, leading to E-cadherin degradation and EMT induction in colorectal epithelial cells.

S100P is a molecular determinant of E-cadherin function in gastric cancer

BackgroundE-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer.MethodsA data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA).ResultsS100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad− tumours, despite not being significantly associated with overall survival on its own.ConclusionsWe propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies.Graphical abstract

Abstract TMIM-084: PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS

Abstract PURPOSE: Metastasis and carcinomatosis remains a major problem in ovarian cancer. Metastasis requires a process known as Epithelial-to-Mesenchymal Transition (EMT), where by epithelial cancer cells undergo dynamic changes to acquire a mesenchymal and invasive phenotype. Twist1 is a transcription factor with a central role in EMT. We previously showed that in epithelial ovarian cancer (EOC), Twist1 is constitutively ubiquitinated and targeted for proteasomal degradation and inhibition of Twist degradation is associated with mesenchymal phenotype and chemoresistance. Accordingly, identification of mechanisms that promote EMT by inhibiting the active degradation of Twist1 is critical for prevention of carcinomatosis. In this study, we report the identification of PKCα as a central regulator of Twist1. We identified 11 possible PKCα phosphorylation sites on Twist1. We show that Twist1 is a novel substrate of the kinase PKCα and that PKCα-induced Twist1 phosphorylation abrogates Twist1 ubiquitination leading to its stabilization and consequently EMT. METHODS: The following cell lines were used: (1) ovarian cancer cells lines OVCAR3 and OVCA432; (2) in-house developed cultures of ovarian cancer cells, R182 and R2615; and (3) HEK293T cells. The effect of constitutively active (PKCαCAT), dominant negative (PKCαDN), and wild-type (wt-PKCα) PKCα on levels of Twist1 were determined by transient transfection using qRT-PCT and western blot analysis. Levels of phosphorylated Twist1 were measured in Twist1 immunoprecipitate-complex using anti-phospho-serine/threonine/tyrosine antibody. The phospho-deficient mutant Twist1 S144A was constructed using QuickChange Site-directed Mutagenesis Kit. PKCα was knocked-out in ovarian cancer cells using CRISPR/Cas9. EMT was induced by treatment with 10 ng/ml TGFβ1 and confirmed molecularly (i.e. loss of epithelial markers E-cadherin, Ck18, Claudin 3 and gain of mesenchymal markers Twist1, N-cadherin, and vimentin). Intra-peritoneal tumors were established by injecting 10 million ovarian cancer cells in athymic nude mice. RESULTS: Transfection with PKCαCAT(active) resulted in a significant increase in Twist1 protein levels compared to PKCαDN(inactive) or empty vector control in EOC and HEK293T cells. This was not a transcriptional effect (no increase in Twist1 mRNA) but due to increase in the levels of phospho-serine/threonine/tyrosine on Twist1. We identified S144 as the putative PKCα phosphorylation site on Twist1 protein. Whereas wt-Twist1 was readily ubiquitinated when transfected in ovarian cancer cells and HEK293T, the phosphomimic S144D Twist1 demonstrated significantly less ubiquitination. Furthermore, we identified TGFβ1 as an activator of the endogenous PKCα-Twist1 axis in ovarian cancer cells. TGFβ1 is able to: (1) activate PKCα; (2) increase Twist1 protein levels; (3) and induce EMT (spheroid formation). Knock-out of PKCα in ovarian cancer cells abrogated TGFβ1-induced EMT in vitro and inhibit carcinomatosis in vivo in athymic nude mice model. CONCLUSION: We demonstrate for the first time a TGFβ1-PKCα-Twist1 signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization. This is a non-classical pathway of TGFβ1 induced EMT. Moreover, we identify S144 on Twist1 as novel and direct PKCα-phosphorylation site that can control Twist1 stability. Given the pleiotropic nature of TGFβ1 signaling, the identification of PKCα as a novel target may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation in ovarian cancer. Citation Format: Roslyn Tedja, Cai Roberts, Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Yang Yang-Hartwich, Carlotta Glackin and Gil G. Mor. PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-084.

Cellular senescence and EMT crosstalk in bleomycin-induced pathogenesis of pulmonary fibrosis-an in vitro analysis.

With poor prognosis and aberrant lung remodeling, pulmonary fibrosis exhibits worldwide prevalence accompanied by an increase in burden in terms of hospitalization and death. Apart from genetic and non-genetic factors, fibrosis occurs as a side effect of bleomycin antineoplastic activity. Elucidating the cellular and molecular mechanism could help in the development of effective anti-fibrotic treatment strategies. In the present study, we investigated the underlying mechanism behind bleomycin-induced fibrosis using human alveolar epithelial cells (A549 cells). On the basis of the experimental observation, it was demonstrated that with transforming growth factor-β (TGF-β) as a central mediator of fibrosis progression, a cross-talk between epithelial-mesenchymal transition (EMT) and senescence upon bleomycin treatment occurs. This results in the advancement of this serious fibrotic condition. Fibrosis was initiated through integrin activation and imbalance in the redox state (NOX expression) of the cell. It progressed along the TGF-β-mediated non-canonical pathway (via ERK phosphorylation) followed by the upregulation of α-smooth muscle actin and collagen synthesis. Additionally, in this process, the loss of the epithelial marker E-cadherin was observed. Furthermore, the expressions of senescence markers, such as p21 and p53, were upregulated upon bleomycin treatment, thereby intensifying the fibrotic condition. Accordingly, the molecular pathway mediating the bleomycin-induced fibrosis was explored in the current study.

The Effect of Interval Training on the Expression of Tumor Suppressor Gene, Systemic Inflammation, and Tumor Volume in Breast Cancer–Bearing BALB/c Mice

Introduction: E-cadherin is expressed in most normal epithelial tissues. Loss of E-cadherin can cause dedifferentiation and invasiveness in human carcinomas, leading E-cadherin to be classified as a tumor suppressor. Therefore, the aim of this study was to investigate the effect of interval training on the expression of tumor suppressor gene E-cadherin in breast cancer-bearing BALB/c mice. Methods: Twenty female BALB/c mice were purchased from the Pasteur Institute and transferred to the Animal Laboratory of Tarbiat Modares University and randomly divided into four groups of training-cancer-rest, training-cancer-training, rest-cancer-rest, and rest-cancer-training. Cancer was induced by subcutaneous injection of 4T1 cell line. The mice performed an average-intensity interval training for 10 weeks, 5 days per week. Forty-eight hours after the last exercise session, the mice were sacrificed to measure the research variables. Gene expression was investigated using real-time PCR. The Kruskal-Wallis test was used to determine the statistical differences between groups. Results: There was a significant difference in the E-cadherin gene expression between the training-cancer-training and rest-cancer-rest groups (p = 0.03). This difference was also observed between the training-cancer-training and training-cancer-rest groups (p = 0.04). Conclusion: Interval exercise training may influence the expression of major tumor suppressor genes and systemic inflammation involved in the development of metastasis and even reverse this process.

The comprehensive role of E-cadherin in maintaining prostatic epithelial integrity during oncogenic transformation and tumor progression.

E-cadherin complexes with the actin cytoskeleton via cytoplasmic catenins and maintains the functional characteristics and integrity of the epithelia in normal epithelial tissues. Lost expression of E-cadherin disrupts this complex resulting in loss of cell polarity, epithelial denudation and increased epithelial permeability in a variety of tissues. Decreased expression of E-cadherin has also been observed in invasive and metastatic human tumors. In this study, we investigated the effect of E-cadherin loss in prostatic epithelium using newly developed genetically engineered mouse models. Deletion of E-cadherin in prostatic luminal epithelial cells with modified probasin promoter driven Cre (PB-Cre4) induced the development of mouse prostatic intraepithelial neoplasia (PIN). An increase in levels of cytoplasmic and nuclear β-catenin appeared in E-cadherin deleted atypical cells within PIN lesions. Using various experimental approaches, we further demonstrated that the knockdown of E-cadherin expression elevated free cytoplasmic and nuclear β-catenin and enhanced androgen-induced transcription and cell growth. Intriguingly, pathological changes representing prostatic epithelial cell denudation and increased apoptosis accompanied the above PIN lesions. The essential role of E-cadherin in maintaining prostatic epithelial integrity and organization was further demonstrated using organoid culture approaches. To directly assess the role of loss of E-cadherin in prostate tumor progression, we generated a new mouse model with bigenic Cdh1 and Pten deletion in prostate epithelium. Early onset, aggressive tumor phenotypes presented in the compound mice. Strikingly, goblet cell metaplasia was observed, intermixed within prostatic tumor lesions of the compound mice. This study provides multiple lines of novel evidence demonstrating a comprehensive role of E-cadherin in maintaining epithelial integrity during the course of prostate oncogenic transformation, tumor initiation and progression.