Reduced E-cadherin Expression Research Articles

Regulatory mechanisms of haptoglobin on particulate matter-induced epithelial-to-mesenchymal transition in bronchial epithelial cells.

It has been proposed that repeated exposure of bronchial epithelial cells to atmospheric particulate matter (PM) could disrupt airway epithelial integrity and lead to epithelial-to-mesenchymal transition (EMT) and ultimately airway remodeling. The molecular mechanisms underlying PM-related bronchial epithelial EMT have not yet been elucidated. The aim of this research is to clarify the molecular mechanism of EMT upon PM exposure. Using an in vivo mouse model of PM-induced airway inflammation and an in vitro model of PM-stimulated bronchial epithelial cells, we clarified the role of haptoglobin (HP) in PM-induced bronchial epithelial EMT. The expression of HP in lung tissues was evaluated by immunohistochemistry (IHC). Western blotting (WB) and immunofluorescence staining were used to analyze EMT-related protein expression and the relevant signaling pathways in the changes in lung tissues and bronchial epithelial cells upon PM exposure. HP small interfering RNA (siRNA) was used to implement the interference of endogenous HP. In vivo experiments showed elevated HP expression in the bronchial epithelium upon PM exposure. IHC and WB showed that E-cadherin expression was decreased, and vimentin expression was increased in bronchial epithelial cells. Moreover, WB results showed that the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular regulated protein kinases (ERK) were elevated in the lung parenchymal tissue of mice. The results of in vitro molecular mechanism experiments showed that compared with those of the control group, the phosphorylation levels of STAT3 and ERK in the PM group increased progressively with higher concentrations of PM and longer stimulation durations. BEAS-2B cells were pretreated with stattic (STAT3 inhibitor) and/or U0126 (ERK inhibitor), and it was found that either stattic or U0126 inhibited PM-induced reduction of E-cadherin expression and elevation of vimentin expression, and the inhibitory effect was most significant when both inhibitors were pretreated simultaneously. Through transfection of BEAS-2B cells with HP siRNA, WB results showed that HP siRNA partially reversed the PM-induced reduction in E-cadherin expression and elevation of vimentin expression, in addition to the reduction in the phosphorylation levels of the STAT3 and ERK. HP is an important mediator of PM-induced EMT in bronchial epithelial cells and promotes PM-induced EMT in bronchial epithelial cells through activation of the STAT3 signaling pathway and the ERK signaling pathway. Inhibition of HP expression attenuates PM exposure-induced EMT in bronchial epithelial cells.

Relevance of the Immunohistochemical Expression of p53 and E-cadherin in the Grading of Urothelial Carcinoma: A Single-Center Cross-Sectional Observational Study.

Background Urothelial carcinoma (UC) is a prevalent cancer worldwide, primarily affecting the urinary bladder. It is more common in men than women and is often linked to factors like tobacco smoking, occupational exposure, and chronic infections. UC can be classified into different subtypes based on its growth pattern (papillary or non-papillary) and the extent of invasion into the bladder wall. The management of UC depends on its grade and stage, with treatment options ranging from transurethral resection of bladder tumor (TURBT) for non-invasive tumors to cystectomy for muscle-invasive disease. This study aimed to investigate the expression of p53 and E-cadherin in low-grade and high-grade UC and their correlation with histomorphological parameters. Materials and methods A cross-sectional observational study was conducted on 50 histopathologically confirmed UC cases. Immunohistochemical (IHC) staining for p53 and E-cadherin was performed, and the results were correlated with clinicopathological features. Statistical analysis was performed to find the correlation between p53 and E-cadherin immunoexpression and the grade of UC. Results The cohort's mean age was 58.18±12.99 years, with a male predominance (76%). Most cases (52%) were from rural areas and unskilled workers (52%). Hematuria was present in all cases (100%), while urgency and frequency were reported in 32% each. High-grade UC was more common (76%), with 48% being invasive. Muscle invasion was absent in 56%. p53 overexpression was seen in all cases, with 38% moderate and 36% strong staining. Twenty-two out of 24 cases (91.7%) of invasive UC showed low E-cadherin expression, while all 26 cases (100%) of low-grade papillary UC and high-grade papillary UC displayed high E-cadherin expression. This suggests that reduced E-cadherin expression is strongly associated with invasive UC, potentially as a tumor aggressiveness and progression marker. Conclusion Considering that elevated p53 protein expression is linked to aggressive tumor behavior, a more intensive treatment strategy is recommended for patients with non-muscle-invasive bladder cancer (NMIBC) who show high p53 protein scores. On the other hand, the downregulation or absence of E-cadherin expression has been recognized as a strong indicator of advanced grade and higher clinical stages.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

ELP1, the Gene Mutated in Familial Dysautonomia, Is Required for Normal Enteric Nervous System Development and Maintenance and for Gut Epithelium Homeostasis.

Familial dysautonomia (FD) is a rare sensory and autonomic neuropathy that results from a mutation in the ELP1 gene. Virtually all patients report gastrointestinal (GI) dysfunction and we have recently shown that FD patients have a dysbiotic gut microbiome and altered metabolome. These findings were recapitulated in an FD mouse model and moreover, the FD mice had reduced intestinal motility, as did patients. To understand the cellular basis for impaired GI function in FD, the enteric nervous system (ENS; both female and male mice) from FD mouse models was analyzed during embryonic development and adulthood. We show here that not only is Elp1 required for the normal formation of the ENS, but it is also required in adulthood for the regulation of both neuronal and non-neuronal cells and for target innervation in both the mucosa and in intestinal smooth muscle. In particular, CGRP innervation was significantly reduced as was the number of dopaminergic neurons. Examination of an FD patient's gastric biopsy also revealed reduced and disoriented axons in the mucosa. Finally, using an FD mouse model in which Elp1 was deleted exclusively from neurons, we found significant changes to the colon epithelium including reduced E-cadherin expression, perturbed mucus layer organization, and infiltration of bacteria into the mucosa. The fact that deletion of Elp1 exclusively in neurons is sufficient to alter the intestinal epithelium and perturb the intestinal epithelial barrier highlights a critical role for neurons in regulating GI epithelium homeostasis.

Morphine promotes non-small cell lung cancer progression by downregulating E-cadherin via the PI3K/AKT/mTOR pathway

Morphine has been suggested to affect cancer cell dynamics and decrease survival rates in lung cancer patients at specific doses, but the precise mechanisms poorly understood. In this study, we aimed to investigate the molecular mechanisms by which morphine modulates the malignant characteristics of non-small cell lung cancer. Cell proliferation was assessed via the Cell Counting Kit-8 assay, and cell migration and invasion were examined via wound healing and Transwell assays. We employed immunofluorescence staining to evaluate E-cadherin expression in A549 and Lewis lung cancer (LLC) cell lines and immunohistochemistry to evaluate E-cadherin expression in nude mice tumours. Additionally, the in vivo effects of morphine on lung cancer progression were explored in a xenograft tumour experiments, in which naloxone was used as a morphine antagonist. Western blot analysis was performed to detect E-cadherin, phosphorylated mTOR (p-mTOR), mTOR, phosphorylated AKT (p-AKT), AKT, phosphorylated PI3K (p-PI3K), and PI3K protein levels in A549 and LLC cells as well as in tumour samples. Morphine (10 µM) significantly increased the proliferation of A549 and LLC cells in vitro (p < 0.05). It also enhanced the migratory and invasive capacities of these cell lines (p < 0.01). Mechanistically, morphine treatment (10 µM) led to a reduction in the expression of E-cadherin, and an increase in the phosphorylation of PI3K, AKT, and mTOR in A549 and LLC cells (p < 0.01). Morphine treatment (1.5 mg/kg) also reduced E-cadherin expression in xenograft tumours and promoted tumour growth in vivo (p < 0.05). This effect was reversed by naloxone (0.1 mg/kg). The results demonstrated that morphine stimulates the malignant proliferation of A549 and LLC cell lines and promotes xenograft tumour growth. Perhaps by specifically targeting MOR, morphine triggers a signalling cascade that activates the PI3K/AKT/mTOR pathway while inhibiting the EMT marker E-cadherin, which may consequently promote the progression of lung cancer.

Spheroid Model of Mammary Tumor Cells: Epithelial-Mesenchymal Transition and Doxorubicin Response.

Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial-mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial-mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored.

Reduced expression of E-cadherin correlates with poor prognosis and unfavorable clinicopathological features in gastric carcinoma: a meta-analysis.

Gastric carcinoma (GC) is one of the most fatal human malignancies globally, with a median survival time less than 1 year. E-cadherin exerts a crucial role in the development and progression of GC as an adhesive, invasive suppressor gene. Whether reduced E-cadherin has an impact on prognosis, clinicopathological features for GC has been well studied, but no conclusive results has been obtained. Eligible studies and relevant data were obtained from PubMed, Elsevier, Embase, Cochrane Library and Web of Science databases until June 30, 2023. A fixed- or random-effects model was used to calculate pooled odds ratios (OR) and 95% confidence intervals (CI). Correlation of E-cadherin expression with overall survival (OS), clinicopathological features and risk factors were evaluated. 36 studies fulfilled the selected criteria. 9048 cases were included. This meta-analysis showed that patients with GC with reduced E-cadherin had unfavourable clinicopathological features and poor OS. The pooled ORs of one-, three- and five-year OS were 0.38 (n = 25 studies, 95%CI: 0.25-0.57, Z = 4.61, P < 0.00001), 0.33 (n = 25 studies, 95% CI: 0.23-0.47, Z = 6.22, P < 0.00001), 0.27 (n = 22 studies, 95% CI: 0.18-0.41, Z = 6.23, P < 0.00001), respectively. Moreover, reduced E-cadherin expression significantly correlated with differentiation grade (OR = 0.29, 95% CI: 0.22-0.39, Z = 8.58, P < 0.00001), depth of invasion (OR = 0.49, 95% CI: 0.36-0.66, Z = 4.58, P < 0.00001), lymphatic node metastasis (OR = 0.49, 95% CI: 0.38-0.64, Z = 5.38, P < 0.00001), distant metastasis (OR = 2.24, 95% CI: 1.62-3.09, Z = 4.88, P < 0.00001), peritoneal metastasis (OR = 2.17, 95% CI: 1.39-3.39, Z = 3.40, P = 0.0007), TNM stage (OR = 0.41, 95% CI: 0.28-0.61, Z = 4.44, P < 0.00001), lymphatic vessel invasion (OR = 1.77, 95% CI: 1.11-2.82, Z = 2.39, P = 0.02), vascular invasion (OR = 1.55, 95% CI: 1.22-1.96, Z = 3.58, P = 0.0003), Lauren type (OR = 0.35, 95% CI: 0.21-0.57, Z = 4.14, P < 0.0001), Borrmann classification (OR = 0.50, 95% CI: 0.25-0.99, Z = 1.97, P = 0.048) and tumor size (≥5 cm vs. <5 cm: OR = 1.73, 95% CI: 1.34-2.23, Z = 4.19, P < 0.0001; ≥6 cm vs. <6 cm: OR = 2.29, 95% CI: 1.51-3.49, Z = 3.87, P = 0.0001). No significant association was observed between reduced E-cadherin expression and liver metastasis, perineural invasion, alcohol consumption, smoking status, familial history, Helicobacter pylori (HP) infection. The reduced expression of E-cadherin is significantly correlated with poor OS and unfavourable clinicopathological features in GC. The expression level of E-cadherin not only serves as a predictor for disease progression and prognosis in GC but also emerges as a novel therapeutic target.

Expression of DARS2 in colorectal cancer and its clinical significance

Objective: To investigate the expression of DARS2 and its clinical significance in colorectal cancer. Methods: In this study, bioinformatics tools, especially gene expression profile interactive analysis 2 (GEPIA2), were used to conduct an in-depth analysis of DARS2 expression in colorectal cancer tissues. Immunohistochemical staining was carried out in 108 colorectal cancer specimens and 30 normal colorectal tissues obtained from the First Affiliated Hospital of Nanchang University, Nanchang, China. Colorectal cancer cell lines (HCT116 and SW480) were transfected with small interfering RNA (siRNA) and DARS2 overexpression plasmid to examine the effects of DARS2 knockdown and overexpression on cell function. To assess the effects on cell function, CCK8 and transwell migration assays were used to assess proliferation and cell motility, respectively. Additionally, protein immunoblotting was employed to scrutinize the expression of proteins associated with the epithelial-mesenchymal transition of colorectal cancer cells. Results: DARS2 exhibited a pronounced upregulation in expression within colorectal cancer tissues compared to their normal epithelial counterparts. Furthermore, DARS2 expression was higher in colorectal cancer of stage Ⅲ-Ⅳ than those of stage Ⅰ-Ⅱ, exhibiting a significant correlation with N staging, M staging, and pathological staging (P<0.05). Kaplan-Meier analyses showed a decreased overall survival rate in colorectal cancer with DARS2 expression compared to those without DARS2 expression (P<0.05). In the siRNA transfection group, there was a significant reduction in cell proliferation and migration (P<0.01 and P<0.05, respectively). Conversely, the transfection of DARS2 overexpression plasmids substantially increased both cell proliferation and migration (P<0.05). Additionally, immunoblotting revealed that DARS2 knockdown led to an upregulation of E-cadherin expression and a downregulation of N-cadherin and vimentin expression. In contrast, DARS2 overexpression resulted in increased N-cadherin and vimentin expression, coupled with reduction in E-cadherin expression. Conclusions: There is a strong association between DARS2 expression and colorectal cancer progression. Silencing DARS2 inhibits cell proliferation and migration, exerting a discernible influence on the epithelial-mesenchymal transition process.

IGF-1R, E-cadherin, N-cadherin, and occludin gene expression and resistance to EGFR tyrosine-kinase inhibitors in patients with lung adenocarcinoma in Serbia.

8524 Background: Mutations in epidermal growth factor-receptor ( EGFR) are found in approximately 10% of patients with lung adenocarcinoma in Serbia. EGFR tyrosine-kinase inhibitors ( EGFR-TKIs) is a safe and effective targeted therapy. However, most patients develop resistance. Acquired resistance to EGFR-TKI is defined as disease progression after achieving response or stable disease and there is a possible molecular association with epithelial-mesenchymal transition (EMT), particularly N-cadherin, E-cadherin, occludin and insulin-like growth factor-1 receptor (IGF-1R). We assessed the gene expression levels of IGF-1R, E-cadherin, N-cadherin, and occludin as predictive factors of resistance to EGFR-TKIs and survival outcomes in patients with advanced EGFR mutation-positive lung adenocarcinoma in Serbia. Methods: 101 patients with stage IIIB/IV EGFR mutation positive lung adenocarcinoma treated with EGFR-TKIs were included in the study. Quantitative RT-PCR was performed to determine mutations in EGFR, and expression of N-cadherin, E-cadherin, occludin and IGF-1R. Kaplan-Meier method was employed to estimate overall-survival (OS) and progression-free survival (PFS), with significance set at p&lt;0.05. Results: Median age of patients was 61.7 (39 - 81) years, 65.3% were females and 91% were diagnosed with metastatic lung adenocarcinoma. EGFR mutations were more frequently detected in females (p &lt;0.001). Median overall survival (OS) was 21 months (15.2–26.7 CI 95%) and progression-free survival (PFS) was 10 months (7.4-12.5 CI 95%). Patients whose tumors had lower levels of IGF-1R, and N-cadherin expression had statistically significantly longer PFS (p &lt;0.001). Patients with lower levels of IGF-1R and higher levels of E-cadherin gene expression were more likely to have a complete or partial response to therapy (p = 0.04). There was a statistically significant association between elevated IGF-1R expression and the occurrence of EGFR-TKI resistance in patients (p &lt;0.001). There was a statistically significant association between increased N-cadherin expression and reduced E-cadherin expression with EGFR-TKI resistance in patients. Conclusions: The hallmark of EMT is the upregulation of N-cadherin followed by the downregulation of E-cadherin. In this group of patients, higher levels of IGF-1R and N-cadherin gene expression were associated with shorter PFS and resistance to EGFR TKI, compared to higher levels of expression of E-cadherin, which may indicate potential value of expression of these genes in predicting acquired resistance to EGFR-TKI. Therefore, more research is needed to prove the potential predictive value of E-cadherin, N-cadherin and IFG-1R in acquired resistance to EGFR-TKI and developing novel therapeutic options with anti-EMT properties that can overcome this problem.

A bioprinted sea-and-island multicellular model for dissecting human pancreatic tumor-stroma reciprocity and adaptive metabolism

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its intricate microenvironment characterized by desmoplasia and complex tumor-stroma interactions. Conventional models hinder studying cellular crosstalk for therapeutic development. To recapitulate key features of PDAC masses, this study creates a novel sea-and-island PDAC tumor construct (s&i PTC). The s&i PTC consists of 3D-printed islands of human PDAC cells positioned within an interstitial extracellular matrix (ECM) populated by human cancer-associated fibroblasts (CAFs). This design closely mimics the in vivo desmoplastic architecture and nutrient-poor conditions. The model enables studying dynamic tumor-stroma crosstalk and signaling reciprocity, revealing both known and yet-to-be-discovered multicellular metabolic adaptations. Using the model, we discovered the orchestrated dynamic alterations of CAFs under nutrient stress, resembling critical in vivo human tumor niches, such as the secretion of pro-tumoral inflammatory factors. Additionally, nutrient scarcity induces dynamic alterations in the ECM composition and exacerbates poor cancer cell differentiation—features well-established in PDAC progression. Proteomic analysis unveiled the enrichment of proteins associated with aggressive tumor behavior and ECM remodeling in response to poor nutritional conditions, mimicking the metabolic stresses experienced by avascular pancreatic tumor cores. Importantly, the model's relevance to patient outcomes is evident through an inverse correlation between biomarker expression patterns in the s&i PTCs and PDAC patient survival rates. Key findings include upregulated MMPs and key ECM proteins (such as collagen 11 and TGFβ) under nutrient-avid conditions, known to be regulated by CAFs, alongside the concomitant reduction in E-cadherin expression associated with a poorly differentiated PDAC state under nutrient deprivation. Furthermore, elevated levels of hyaluronic acid (HA) and integrins in response to nutrient deprivation underscore the model's fidelity to the PDAC microenvironment. We also observed increased IL-6 and reduced α-SMA expression under poor nutritional conditions, suggesting a transition of CAFs from myofibroblastic to inflammatory phenotypes under a nutrient stress akin to in vivo niches. In conclusion, the s&i PTC represents a significant advancement in engineering clinically relevant 3D models of PDAC masses. It offers a promising platform for elucidating tumor-stroma interactions and guiding future therapeutic strategies to improve patient outcomes.

CSChighE-cadherinlow immunohistochemistry panel predicts poor prognosis in oral squamous cell carcinoma

Identifying marker combinations for robust prognostic validation in primary tumour compartments remains challenging. We aimed to assess the prognostic significance of CSC markers (ALDH1, CD44, p75NTR, BMI-1) and E-cadherin biomarkers in OSCC. We analysed 94 primary OSCC and 67 metastatic lymph node samples, including central and invasive tumour fronts (ITF), along with clinicopathological data. We observed an increase in ALDH1+/CD44+/BMI-1- tumour cells in metastatic lesions compared to primary tumours. Multivariate analysis highlighted that elevated p75NTR levels (at ITF) and reduced E-cadherin expression (at the tumour centre) independently predicted metastasis, whilst ALDH1high exhibited independent predictive lower survival at the ITF, surpassing the efficacy of traditional tumour staging. Then, specifically at the ITF, profiles characterized by CSChighE-cadherinlow (ALDH1highp75NTRhighE-cadherinlow) and CSCintermediateE-cadherinlow (ALDH1 or p75NTRhighE-cadherinlow) were significantly associated with worsened overall survival and increased likelihood of metastasis in OSCC patients. In summary, our study revealed diverse tumour cell profiles in OSCC tissues, with varying CSC and E-cadherin marker patterns across primary tumours and metastatic sites. Given the pivotal role of reduced survival rates as an indicator of unfavourable prognosis, the immunohistochemistry profile identified as CSChighE-cadherinlow at the ITF of primary tumours, emerges as a preferred prognostic marker closely linked to adverse outcomes in OSCC.

Regulatory role of lncRNA MALAT1 and miR-150-3p interaction in breast cancer progression and therapeutic implications

Long non-coding RNAs (lncRNAs) play crucial roles in cancer progression and metastasis. However, the precise regulatory mechanisms of MALAT1, an lncRNA, in breast cancer remain elusive. Consequently, this study explored the specific role of MALAT1 as a tumor promoter in breast cancer. Initially, MALAT1-small interfering RNA (siRNA) transfection resulted in the inhibition of breast cancer cell migration, colony formation, and invasion. Real-time polymerase chain reaction analysis also revealed increased E-cadherin expression and decreased vimentin and vascular endothelial growth factor (VEGF) mRNA levels subsequent to the transfection. Bioinformatic analysis further uncovered a specific interaction between MALAT1 and miR-150-3p, indicating elevated MALAT1 expression in breast cancer with a negative correlation to miR-150-3p expression. Furthermore, the overexpression of MALAT1 markedly suppressed miR-150-3p expression, indicating a reciprocal inhibition between the two. Luciferase reporter assays confirmed the specific association between miR-150-3p and MALAT1 at the sequence level. Furthermore, transfection with miR-150-3p mimic inhibited breast cancer cell migration, colony formation, and invasion, along with increased E-cadherin expression and decreased Vimentin and VEGF mRNA levels. Conversely, miR-150-3p inhibitor transfection led to opposing effects, reducing E-cadherin expression, and elevating vimentin and VEGF mRNA levels, while also inhibiting migration, colony formation, and invasion. Functionally, MALAT1 siRNA restrained breast cancer cell proliferation and migration while enhancing cellular apoptosis on administration to breast cancer cells, primarily mediated through miR-150-3p. Our findings delineate MALAT1 as a tumor growth-promoting gene antagonized by miR-150-3p. Building on these insights, this study proposes a potential therapeutic avenue for breast cancer treatment.

Abstract 202: Spry1 targeting enhances E-cadherin expression in cutaneous melanoma

Abstract Background: Cutaneous melanoma (CM) is a very aggressive malignancy that still represents the deadliest form of skin cancer. About 50% of CM harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently reported that Spry1 knockdown (Spry1KO) reduced the expression of several markers of epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Loss of E-cadherin is a hallmark of EMT in CM, and the presence of aberrant E-cadherin expression in CM is usually associated with a significantly worse overall survival. Based on these premises, in this study we wondered to explore whether Spry1 influences EMT through the modulation of E-cadherin expression. Material and Methods: SPRY1 gene was knocked-out using the CRISPR strategy in BRAF wild-type (wt) and in BRAF-mutant CM cell lines. By using in vitro and in vivo models, the effects of Spry1KO on E-cadherin expression and localization was investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass spectrometry (IP-MS) was performed. Results and Discussion: E-cadherin mRNA and protein levels were significantly increased both in vitro and in vivo in Spry1KO clones. In addition, immunofluorescence analysis revealed a sustained redistribution of E-cadherin to the plasma membrane following Spry1 loss, thus confirming that Spry1 plays a role in EMT in CM. E-cadherin requires p120-catenin (p120) to maintain cell adhesion and functional adherens junctions. Indeed, p120 dysregulation results in the rapid turnover of E-cadherin complexes. Intriguingly, IP-MS analyses identified several Spry1 protein partners, including p120. Although a number of functional studies are still ongoing, preliminary western blot analysis of cytosolic and mitochondrial proteins indicated that Spry1 and p120 were mostly located in mitochondria. Conclusion: Spry1 reduced E-cadherin expression in vitro and in vivo. Starting from preliminary data, we hypothesized that Spry1-p120 interaction might influence E-cadherin distribution over the plasma membrane. Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula Anneli Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 targeting enhances E-cadherin expression in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 202.

Pro-tumorigenic activity of PYCR1 in gastric cancer through regulating the PI3K/AKT signaling

BackgroundThe primary objective of this investigation was to assess the impact of pyrroline-5-carboxylate reductase 1 (PYCR1) on the progression of gastric cancer (GC), specifically focusing on tumor growth and metastatic potential. MethodsSurgical specimens from patients with different stages of GC were assayed for PYCR1 expression using immunohistochemistry. PYCR1 expression was manipulated by depletion or overexpression approaches in GC cells, and these cells were applied to explore the functional roles of PYCR1. Expression of apoptosis– and metastasis–related markers was quantified through quantitative real-time PCR and Western blot. ResultsHigher PYCR1 expression was ascertained in surgical specimens from patients with GC as compared to noncancerous adjacent tissues. Additionally, PYCR1 overexpression in GC tissues was linked to adverse clinical outcomes. The depletion of PYCR1 in GC cells resulted in a pronounced reduction in proliferation, the induction of apoptosis, and the attenuation of invasion and metastasis. Conversely, its ectopic expression notably augmented proliferation, restricted apoptosis, and stimulated invasion and metastasis. In addition, the knockdown of PYCR1 resulted in a significant elevation in the activation of caspase 3, a key protein involved in apoptosis. This depletion also led to a decrease in the activation or expression of proteins associated with metastasis, such as phosphorylated (p)-phosphatidylinositol 3-kinase (PI3K), p-AKT serine/threonine kinase (AKT), and snail family transcriptional repressor 1 (Snail). Additionally, it resulted in an upregulation of E-cadherin expression. Conversely, the overexpression of PYCR1 notably increased the levels of p-PI3K, p-AKT, and Snail, while simultaneously reducing E-cadherin expression. ConclusionPYCR1, by activating PI3K/AKT signaling, assumes a crucial role in governing malignant characteristics of GC cells, including proliferation, apoptosis, and metastasis. These findings underscore the promising potential of PYCR1 as a diagnostic biomarker and a target for tailored therapeutic interventions in patients with GC.

Krüppel-like factor 4 modulates the miR-101/COL10A1 axis to inhibit renal fibrosis after AKI by regulating epithelial–mesenchymal transition

Acute kidney injury (AKI) can progress to renal fibrosis and chronic kidney disease (CKD), which reduces quality of life and increases the economic burden on patients. However, the molecular mechanisms underlying renal fibrosis following AKI remain unclear. This study tested the hypothesis that the Krüppel-like factor 4 (KLF4)/miR-101/Collagen alpha-1X (COL10A1) axis could inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis after AKI in a mouse model of ischemia-reperfusion (I/R)-induced renal fibrosis and HK-2 cells by gene silencing, overexpression, immunofluorescence, immunohistochemistry, real-time quantitative PCR, Western blotting, dual-luciferase reporter assay, fluorescence in situ hybridization (FISH) and ELISA. Compared with the Sham group, I/R induced renal tubular and glomerular injury and fibrosis, and increased the levels of BUN, serum Scr and neutrophil gelatinase-associated lipocalin (NGAL), Col10a1 and Vimentin expression, but decreased E-cadherin expression in the kidney tissues of mice at 42 days post-I/R. Similarly, hypoxia promoted fibroblastic morphological changes in HK-2 cells and enhanced NGAL, COL10A1, Vimentin, and α-SMA expression, but reduced E-cadherin expression in HK-2 cells. These pathological changes were significantly mitigated in COL10A1-silenced renal tissues and HK-2 cells. KLF4 induces miR-101 transcription. More importantly, hypoxia upregulated Vimentin and COL10A1 expression, but decreased miR-101, KLF4, and E-cadherin expression in HK-2 cells. These hypoxic effects were significantly mitigated or abrogated by KLF4 over-expression in the HK-2 cells. Our data indicate that KLF4 up-regulates miR-101 expression, leading to the downregulation of COL10A1 expression, inhibition of EMT and renal fibrosis during the pathogenic process of I/R-related renal fibrosis.

Abstract C101: Calcium signaling rewiring activates epithelial-to-mesenchymal transition

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States and is often not diagnosed until it has already metastasized. Calcium and downstream signaling cascades contribute to epithelial-to-mesenchymal transition (EMT) and facilitate metastasis formation in PDAC. Ca2+/calmodulin-dependent kinase-II (CaMK2), a downstream effector kinase activated by Ca2+, has previously been shown in vitro to be an important mediator of a partial/hybrid EMT program that gives tumor cells high metastatic proclivity. We hypothesize that CaMK2 is a critical node in the partial EMT state and thus deleted Camk2b in vivo to study the effect of its loss on tumor progression and metastasis. Methods: We generated a genetically engineered mouse model where Camk2b was knocked out in the KPCY autochthonous PDAC mouse model (herein KPCY-Camk2bcKO). Isolated cell lines were treated with the EMT-inducing ligand TGF-b to study the effect of Camk2b on tumor cell plasticity. Results: Prior in vitro work indicated that CAMK2 is required for hybrid EMT and thus we expected KPCY-Camk2bcKO mice to have fewer metastases and live longer. Surprisingly, the genetic deletion of Camk2b accelerated tumor formation and reduced overall survival relative to KPCY controls. Despite dying sooner, there was no difference in overall tumor burden in KPCY-Camk2bcKO mice. Pathological and immunofluorescence analysis of KPCY-Camk2bcKO tumors showed an increased number of poorly differentiated regions and EMT as indicated by loss of the epithelial cell marker ECADHERIN (E-CAD) in YFP lineage-labeled tumor cells. In stark contrast to prior work, the loss of Camk2b increased tumor cell plasticity, potentially highlighting a rewiring of calcium signaling circuits in the Camk2b-null state. To this end, KPCY-Camk2bcKO tumor cell lines displayed a baseline mesenchymal morphology in vitro and had lower expression of Cdh1 (the gene encoding E-CAD) mRNA. Using a flow cytometry-based approach, we also found baseline E-CAD protein levels to be diminished relative to KPCY lines. Additionally, KPCY-Camk2bcKO tumor cells were more responsive to the EMT-inducing ligand TGF-b and downregulated their E-CAD levels more than KPCY cells. Notably, treatment with low dose TGF-b also dramatically reduced E-CAD expression in KPCY-Camk2bcKO but not KPCY cells, indicating that the knockout cells are easily pushed towards a mesenchymal state. Conclusions: While CAMK2 has been studied previously as a facilitator of the EMT program, this work paradoxically demonstrates that Camk2b loss enhances cellular plasticity within PDAC. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive EMT. In future studies, we will investigate the effect of Camk2b loss on the metastatic cascade and uncover mechanisms through which Camk2b loss makes PDAC tumor cells more plastic. We will also begin to characterize the non-cell autonomous roles of CAMK2B in crosstalk with the tumor microenvironment. Citation Format: Jessica Peura, Yamini Ogoti, Jason Pitarresi. Calcium signaling rewiring activates epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C101.

Immunohistochemical Expression Status of HER2/neu and E-cadherin in Prostate Adenocarcinoma: A Cross-sectional Study

Introduction: Prostate cancer is a common tumour, accounting for 92% of malignancies in the male genital tract. Due to the high recurrence rate, it is important to identify prognostic markers in prostate cancer. Human Epidermal Growth Factor Receptor 2 (HER2) and Epithelial cadherin (E-cadherin) are speculated to be associated with carcinogenesis, but the literature has provided controversial results. Aim: To investigate the expression of HER2/neu and E-cadherin in prostatic adenocarcinoma and its correlation with clinicopathological parameters. Materials and Methods: A retrospective cross-sectional study was conducted at the Department of Pathology, SDM University, Dharwad, Karnataka, India. The study duration was one year and five months, from October 2020 to March 2022. A total of 45 cases diagnosed as prostatic adenocarcinoma between October 2016 and September 2019 were included. Clinicopathological parameters such as age, Prostate- Specific Antigen (PSA) levels, metastasis, perineural invasion, Gleason score, Gleason grade, and two-year survival were retrieved from hospital records and through telephone interviews. Immunohistochemistry (IHC) staining for HER2 and E-cadherin was performed and assessed by pathologists. Statistical analysis was conducted using Statistical Package for Social Sciences (SPSS) version 20.0, and the Chi-square test was applied. Results: The mean age of the study participants was 69.6 years, ranging from 52 to 95 years. The rates of HER2/neu overexpression/positivity and reduced E-cadherin expression were 25 (55.5%) and 14 (31.1%), respectively. No statistically significant associations were found between HER2 and E-cadherin expression and age, PSA levels, perineural invasion, Gleason score, Gleason grade, and two-year survival (p&gt;0.05). However, a statistically significant association was observed between HER2 expression and distant metastasis (p-value=0.006), whereas no significant association was found with E-cadherin (p-value=0.81). Conclusion: The study demonstrated a significant association between HER2/neu overexpression and tumour metastasis, suggesting that HER2/neu could serve as a prognostic parameter to assist surgeons. In contrast, E-cadherin showed no association with clinicopathological parameters, raising questions about its involvement in the progression of prostatic adenocarcinoma.

Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

Sphingolipid paracrine signaling impairs keratinocyte adhesion to promote melanoma invasion

Melanoma is the deadliest form of skin cancer due to its propensity to metastasize. It arises from melanocytes, which are attached to keratinocytes within the basal epidermis. Here, we hypothesize that, in addition to melanocyte-intrinsic modifications, dysregulation of keratinocyte functions could initiate early-stage melanoma cell invasion. We identified the lysolipid sphingosine 1-phosphate (S1P) as a tumor paracrine signal from melanoma cells that modifies the keratinocyte transcriptome and reduces their adhesive properties, leading to tumor invasion. Mechanistically, tumor cell-derived S1P reduced E-cadherin expression in keratinocytes via S1P receptor dependent Snail and Slug activation. All of these effects were blocked by S1P2/3 antagonists. Importantly, we showed that epidermal E-cadherin expression was inversely correlated with the expression of the S1P-producing enzyme in neighboring tumors and the Breslow thickness in patients with early-stage melanoma. These findings support the notion that E-cadherin loss in the epidermis initiates the metastatic cascade in melanoma.

Macrophage migration inhibitory factor exacerbates asthmatic airway remodeling via dynamin-related protein 1-mediated autophagy activation

BackgroundMacrophage migration inhibitory factor (MIF) and GTPase dynamin-related protein 1 (Drp1)-dependent aberrant mitochondrial fission are closely linked to the pathogenesis of asthma. However, it is unclear whether Drp1-mediated mitochondrial fission and its downstream targets mediate MIF-induced proliferation of airway smooth muscle cells (ASMCs) in vitro and airway remodeling in chronic asthma models. The present study aims to clarify these issues.MethodsIn this study, primary cultured ASMCs and ovalbumin (OVA)-induced asthmatic rats were applied. Cell proliferation was detected by CCK-8 and EdU assays. Western blotting was used to detect extracellular signal-regulated kinase (ERK) 1/2, Drp1, autophagy-related markers and E-cadherin protein phosphorylation and expression. Inflammatory cytokines production, airway reactivity test, histological staining and immunohistochemical staining were conducted to evaluate the development of asthma. Transmission electron microscopy was used to observe the mitochondrial ultrastructure.ResultsIn primary cultured ASMCs, MIF increased the phosphorylation level of Drp1 at the Ser616 site through activation of the ERK1/2 signaling pathway, which further activated autophagy and reduced E-cadherin expression, ultimately leading to ASMCs proliferation. In OVA-induced asthmatic rats, MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) treatment, suppression of mitochondrial fission by Mdivi-1 or inhibiting autophagy with chloroquine phosphate (CQ) all attenuated the development of airway remodeling.ConclusionsThe present study provides novel insights that MIF promotes airway remodeling in asthma by activating autophagy and degradation of E-cadherin via ERK/Drp1 signaling pathway, suggesting that targeting MIF/ERK/Drp1 might have potential therapeutic value for the prevention and treatment of asthma.