Adenocarcinoma Invasion Research Articles

Identification of HMGB2 associated with proliferation, invasion and prognosis in lung adenocarcinoma via weighted gene co-expression network analysis

BackgroundHigh mobility group protein B2 (HMGB2) is a multifunctional protein that plays various roles in different cellular compartments. Moreover, HMGB2 serves as a potential prognostic biomarker and therapeutic target for lung adenocarcinoma (LUAD).MethodsIn this study, the expression pattern, prognostic implication, and potential role of HMGB2 in LUAD were evaluated using the integrated bioinformatics analyses based on public available mRNA expression profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases, both at the single-cell level and the tissue level. Further study in the patient-derived samples was conducted to explore the correlation between HMGB2 protein expression levels with tissue specificity, (tumor size-lymph node-metastasis) TNM stage, pathological grade, Ki-67 status, and overall survival. In vitro experiments, such as CCK-8, colony-formation and Transwell assay, were performed with human LUAD cell line A549 to investigate the role of HMGB2 in LUAD progression. Furthermore, xenograft tumor model was generated with A549 in nude mice.ResultsThe results showed that the HMGB2 expression was higher in the LUAD samples than in the adjacent normal tissues and was correlated with high degree of malignancy in different public data in this study. Besides, over-expression of HMGB2 promoted A549 cells proliferation and migration while knocking down of HMGB2 suppressed the tumor promoting effect.ConclusionsOur study indicated that HMGB2 was remarkably highly expressed in LUAD tissues, suggesting that it is a promising diagnostic and therapeutic marker for LUAD in the future.

MiR-579 Inhibits Lung Adenocarcinoma Cell Proliferation and Metastasis via Binding to CRABP2.

Background Lung cancer is the cancer with the highest morbidity and mortality. Lung adenocarcinoma (LUAD) is a subtype of lung cancer. The aim of this study is to explore the functions of miR-579 and CRABP2 in lung adenocarcinoma. Methods Cell counting kit-8 (CCK-8) and colony formation assays were applied to calculate cell proliferative abilities. Transwell assay was utilized to measure cell invasive ability. Results MiR-579 is low expressed in LUAD tissues and cell lines. MiR-579 inhibits cell viability and invasion of lung adenocarcinoma. Knockdown of CRABP2 inhibits cell proliferation and invasion of Calu-3 cells. MiR-579 suppresses cell proliferation and invasion by regulating CRABP2 in Calu-3 cells. Conclusion Our study reveals that miR-579 acts as a tumor suppressor in LUAD and miR-579 can target and regulate the expression of CRABP2 to mediate cell proliferation and invasion. This study indicates that miR-579 has a potential to be a candidate biomarker for the treatment of LUAD.

MiRNA-338-3p inhibits the migration, invasion and proliferation of human lung adenocarcinoma cells by targeting MAP3K2.

Objective: This study aimed to investigate the effects of micro ribonucleic acid (miR)-338-3p on the migration, invasion and proliferation of lung adenocarcinoma (LUAD) cells.Methods: Bioinformatics analysis was employed to evaluate the function and expression of related genes in lung cancer. Human A549 and NCI-H1299 cells cultured to logarithmic growth stage were assigned to negative control (NC) mimic group, miR-338-3p mimic group (miR-mimic group), NC inhibitor group and miR-338-3p inhibitor group (miR-inhibitor group) treated with or without MAP3K2 overexpression (OE)-lentivirus, or TBHQ or FR180204. Transwell assay, cell colony formation assay, Western blotting and cell-cycle analysis were carried out.Results: Bioinformatics results manifested that miR-338 and MAP3K2 were involved in LUAD. The expression levels of MAP3K2, p-ERK1/2, MMP-2, MMP-3, MMP-9, cyclin A2 and cyclin D1 were increased after addition of miR-338-3p inhibitor, consistent with the raised amount of LUAD cells in migration and invasion experiments and number of colonies formed, as well as the cell cycle, but miR-338-3p mimic reversed these results. Moreover, MAP3K2 overexpression elevated the level of p-ERK1/2. Meanwhile, after treatment with TBHQ or FR180204, the influence of miR-338-3p inhibitor or mimic was also verified.Conclusions: MiR-338-3p overexpression can modulate the ERK1/2 signaling pathway by targeting MAP3K2, thus inhibiting the migration, invasion and proliferation of human LUAD cells.

ViSTA: A Novel Network Improving Lung Adenocarcinoma Invasiveness Prediction from Follow-Up CT Series.

Simple SummaryAssessing follow-up computed tomography(CT) series is of great importance in clinical practice for lung nodule diagnosis. Deep learning is a thriving data mining method in medical imaging and has obtained surprising results. However, previous studies mostly focused on the analysis of single static time points instead of the entire follow-up series and required regular intervals between CT examinations. In the current study, we propose a new deep learning framework, named ViSTA, that can better evaluate tumor invasiveness using irregularly serial follow-up CT images to avoid aggressive procedures or delay diagnosis in clinical practice. ViSTA provides a new solution for irregularly sampled data. ViSTA delivers superior performance compared with other static or serial deep learning models. The proposed ViSTA framework is capable of improving performance close to the human level in the prediction of invasiveness of lung adenocarcinoma while being transferrable to other tasks analyzing serial medical data.To investigate the value of the deep learning method in predicting the invasiveness of early lung adenocarcinoma based on irregularly sampled follow-up computed tomography (CT) scans. In total, 351 nodules were enrolled in the study. A new deep learning network based on temporal attention, named Visual Simple Temporal Attention (ViSTA), was proposed to process irregularly sampled follow-up CT scans. We conducted substantial experiments to investigate the supplemental value in predicting the invasiveness using serial CTs. A test set composed of 69 lung nodules was reviewed by three radiologists. The performance of the model and radiologists were compared and analyzed. We also performed a visual investigation to explore the inherent growth pattern of the early adenocarcinomas. Among counterpart models, ViSTA showed the best performance (AUC: 86.4% vs. 60.6%, 75.9%, 66.9%, 73.9%, 76.5%, 78.3%). ViSTA also outperformed the model based on Volume Doubling Time (AUC: 60.6%). ViSTA scored higher than two junior radiologists (accuracy of 81.2% vs. 75.4% and 71.0%) and came close to the senior radiologist (85.5%). Our proposed model using irregularly sampled follow-up CT scans achieved promising accuracy in evaluating the invasiveness of the early stage lung adenocarcinoma. Its performance is comparable with senior experts and better than junior experts and traditional deep learning models. With further validation, it can potentially be applied in clinical practice.

LINC02535/miR-30a-5p/GALNT3 axis contributes to lung adenocarcinoma progression via the NF- κ B signaling pathway

ABSTRACT Long non-coding RNAs (LncRNA) play important roles in multiple types of cancers. We addressed the role of LINC02535 by regulating the miR-30a-5p /GalNAc Transferase 3 (GALNT3) axis to promote the proliferation, migration, and invasion in lung adenocarcinoma (LUAD) cells. The Cancer Genome Atlas (TCGA) database screened differentially expressed lncRNAs. Quantitative real-time PCR analysis (qRT-PCR) confirmed that LINC02535 is highly expressed in LUAD tissues and cells. In vitro experiments showed that LINC02535 promotes the proliferation, migration, and invasion of LUAD cells. A xenograft mouse model was used to show that LINC02535 promotes tumor growth in vivo. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assay results confirmed that LINC02535 targets miR-30a-5p. The Vicia villosa lectin (VVA) pull-down assay indicated that MUC1 is the glycosylation target of GALNT3, and western blot verified that NF-κB is the downstream signaling pathway of MUC1. We found that LINC02535 was increased in LUAD tissues and cells, and LINC02535 was correlated with the poor prognosis of LUAD patients. miR-30a-5p acts as a tumor suppressor in LUAD by targeting GALNT3. We also demonstrated that LINC02535 might function as the sponge of miR-30a-5p to up-regulate GALNT3, and consequently promote the proliferation and metastasis of LUAD. LINC02535 acts as a competing endogenous RNA (ceRNA) to interact with miR-30a-5p, thereby upregulating the expression of GALNT3, enhancing the function of MUC1, and activating the NF-κB signaling pathway, promoting the malignant progression of LUAD cells. Abbreviations: LncRNA:long non-coding RNA; LUAD: lung adenocarcinoma; TCGA: The Cancer Genome Atlas; GALNT3: GalNAc Transferase 3; qRT-PCR: quantitative real-time PCR analysis; RIP: RNA immunoprecipitation; SPF: specific pathogen-free; VVA: Vicia villosa lectin; ceRNA: competing endogenous RNA; MiRNAs: microRNAs; FBS: fetal bovine serum; PBS: Phosphate buffered saline; CCK-8: Cell Counting Kit-8; NSCLC: non-small cell lung cancer; OC: ovarian cancer; HCC: hepatocellular carcinoma.

Computed Tomography Findings for Predicting Invasiveness of Lung Adenocarcinomas Manifesting as Pure Ground-Glass Nodules.

Purpose: To comprehensively evaluate qualitative and quantitative features for predicting invasiveness of pure ground-glass nodules (pGGNs) using multiplanar computed tomography. Methods: Ninety-three resected pGGNs (16 atypical adenomatous hyperplasia [AAH], 18 adenocarcinoma in situ [AIS], 31 minimally invasive adenocarcinoma [MIA], and 28 invasive adenocarcinoma [IA]) were retrospectively included. Two radiologists analyzed qualitative and quantitative features on three standard planes. Univariable and multivariable logistic regression analyses were performed to identify features to distinguish the pre-invasive (AAH/AIS) from the invasive (MIA/IA) group. Results: Tumor size showed high area under the curve (AUC) for predicting invasiveness (.860, .863, .874, and .893, for axial long diameter [AXLD], multiplanar long diameter, mean diameter, and volume, respectively). The AUC for AXLD (cutoff, 11mm) was comparable to that of the volume (P = .202). The invasive group had a significantly higher number of qualitative features than the pre-invasive group, regardless of tumor size. Six out of 59 invasive nodules (10.2%) were smaller than 11mm, and all had at least one qualitative feature. pGGNs smaller than 11mm without any qualitative features (n = 16) were all pre-invasive. In multivariable analysis, AXLD, vessel change, and the presence or number of qualitative features were independent predictors for invasiveness. The model with AXLD and the number of qualitative features achieved the highest AUC (.902, 95% confidence interval .833-.971). Conclusion: In adenocarcinomas manifesting as pGGNs on computed tomography, AXLD and the number of qualitative features are independent risk factors for invasiveness; small pGGNs (<11mm) without qualitative features have low probability of invasiveness.

A comparative study for the evaluation of CT-based conventional, radiomic, combined conventional and radiomic, and delta-radiomic features, and the prediction of the invasiveness of lung adenocarcinoma manifesting as ground-glass nodules

To investigate and compare the performance of conventional, radiomic, combined, and delta-radiomic features to predict the invasiveness of lung adenocarcinoma manifesting as ground-glass nodules (GGNs). The present retrospective study included 216 GGNs confirmed surgically as pulmonary adenocarcinomas. All the thin-section computed tomography (CT) images were imported into the software of the United Imaging Intelligence research portal, and radiomic features were extracted with three-dimensional (3D) regions of interest. Least Absolute Shrinkage and Selection Operator was used to select the optimal radiomic features. Four models were constructed, including conventional, radiomic, combined conventional and radiomic, and delta-radiomic models. The receiver operating characteristic curves were built to evaluate the validity of these. The type, long diameter, shape, margin, vacuole, air bronchus, vascular convergence, and pleural traction exhibited significant differences between pre-invasive lesions (PILs)/minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IA) groups were selected for conventional model building. Nine radiomic features were selected to build the radiomic model. The four models indicated optimal performance (AUC>0.7). The radiomic and combined models exhibited the highest diagnostic efficiency, and their AUC were 0.89 and 0.88 in the training set, and 0.87 and 0.88 in the validation set, respectively. The delta-radiomic model indicated that the AUC was 0.83 in the training set, and 0.76 in the validation set. Finally, the conventional model exhibited an AUC in the training and validation sets of 0.78 and 0.76. The radiomic model and combined model, in particular, and the delta-radiomic model all demonstrated improved diagnostic efficiency in differentiating IA from PIL/MIA than that of the conventional model.

Impact of resection margin status on survival after operation for pancreatic head cancer with extrapancreatic nerve plexus invasion

Extrapancreatic nerve plexus (PL) invasion of pancreatic ductal adenocarcinoma (PDAC) is an important factor for determining resectability and surgical method. We sought to clarify the characteristics of PDAC with PL invasion and clinical impact of the resection margin status on prognosis for PDAC with PL invasion. A total of 242 patients with pancreatic head cancer who underwent pancreatectomy were evaluated. Clinicopathological data and patient survival were analyzed. Pathological PL invasion was observed in 68 patients (28.1%). Patients with PL invasion had significantly shorter disease-free survival (DFS) and showed trends toward worse overall survival (OS) than those without PL invasion. While multivariate analysis revealed that PL invasion was not an independent prognostic factor, PL invasion was associated with extensive venous invasion and a high percentage of lymph node metastases, both of which were independent factors affecting DFS and OS. Among patients with PL invasion, there was no significant difference in DFS and OS between the R0 and R1 resection groups. PL invasion is a common pathological feature of aggressive PDAC with high propensity for invasiveness and metastatic potential. The microscopic resection margin status may not affect the survival of pancreatic head cancer patients with PL invasion.

Relationship between consolidation tumor ratio and tumor-infiltrating lymphocytes in small-sized lung adenocarcinoma.

BackgroundConsolidation tumor ratio (CTR) is associated with cancer progression and histological invasiveness in lung adenocarcinoma (LAD). However, little is known about the association between CTR and immune‐related factors, including tumor‐infiltrating lymphocytes (TILs) density or tumor expression of programmed death ligand 1 (PD‐L1) and indoleamine 2,3‐dioxygenase 1 (IDO1) in small‐sized LAD.MethodsThis study included 258 patients with LAD (<3 cm) who underwent surgery. Patients were assigned to four groups: CTR = 0; 0 < CTR <0.5; 0.5 ≤ CTR <1 (ground‐glass opacity [GGO] group); and CTR = 1 (pure‐solid group). CD4+, CD8+, and FoxP3+ TIL density and PD‐L1 and IDO1 tumor expression were assessed by immunohistochemistry.ResultsAmong the GGO group, CD8+ and FoxP3+ TIL density increased significantly with increasing CTR (p < 0.001 and p < 0.001, respectively). Moreover, PD‐L1 and IDO1 expression was significantly higher in the pure‐solid group than in the GGO group (p < 0.001 and p < 0.001, respectively).ConclusionsCTR was correlated with the abundance of CD8+ and FoxP3+ TILs in the GGO group. PD‐L1 and IDO1 positivity rates were significantly higher in the pure‐solid group than in the GGO group. Increased CTR may be correlated with immunosuppressive condition.

ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38/ATF-2 signalling pathway.

Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ 2 test, Spearman correlation analysis, Kaplan–Meier, log‐rank test, and Cox's proportional hazard model were used to analyse the clinical cases. The effect of ANKRD49 on the LUAD was investigated using CCK‐8, clonal formation, would healing, transwell assays, and nude mice experiment. Expressions of ANKRD49 and its associated downstream protein molecules were verified by real‐time PCR, Western blot, immunohistochemistry, and/or immunofluorescence analyses. ANKRD49 expression was highly elevated in LUAD. The survival rate and Cox's modelling analysis indicated that there may be an independent prognostic indicator for LUAD patients. We also found that ANKRD49 promoted the invasion and migration in both in in vitro and in vivo assays, through upregulating matrix metalloproteinase (MMP)‐2 and MMP‐9 activities via the P38/ATF‐2 signalling pathway Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis and promotes the metastasis of A549 cells via upregulation of MMP‐2 and MMP‐9 in a P38/ATF‐2 pathway‐dependent manner.

Study on the Correlation Between CT Features and Vascular Tumor Thrombus Together With Nerve Invasion in Surgically Resected Lung Adenocarcinoma.

BackgroundWe aimed to analyze the relationship between pulmonary adenocarcinoma patients with vascular tumor thrombus and nerve invasion and different CT features.MethodsThe preoperative CT scanning data of 86 patients with lung adenocarcinoma who underwent surgical resection in our hospital from January 2020 to January 2022 were analyzed in the form of retrospective analysis. The CT images of all patients were observed, and the relationship between them and vascular tumor thrombus and nerve invasion of lung adenocarcinoma was analyzed. At the same time, the sensitivity, specificity, and accuracy of enhanced CT and plain CT were compared to evaluate the diagnostic efficacy of both.ResultsThe results showed that the vascular tumor thrombus of lung adenocarcinoma was mainly related to the solid components and lobulated and calcified tumors in CT images, and the nerve invasion of lung adenocarcinoma was mainly related to the tumors with bronchial inflation sign in CT images (P < 0.05). The sensitivity, specificity, and accuracy of enhanced CT in the diagnosis of vascular tumor thrombus were 78.26%, 96.83%, and 91.86%, respectively, and the sensitivity, specificity, and accuracy in the diagnosis of nerve invasion were 75.00%, 98.72%, and 96.51%, respectively. The sensitivity, specificity, and accuracy of plain CT in the diagnosis of vascular tumor thrombus were 43.48%, 92.06%, and 79.07%, respectively, and the sensitivity, specificity, and accuracy in the diagnosis of nerve invasion were 25.00%, 94.87%, and 88.37%, respectively. The contrast showed that the sensitivity and accuracy of enhanced CT were higher than those of plain CT (P < 0.05), but the difference of specificity was not obvious (P > 0.05).ConclusionsSolid components and lobulated and calcified tumors in CT signs are closely related to vascular tumor thrombus of lung adenocarcinoma, while patients with bronchial inflation sign are related to nerve invasion.

Correlation of computed tomography quantitative parameters with tumor invasion and Ki-67 expression in early lung adenocarcinoma.

The purpose of the study is to investigate the correlation of computed tomography (CT) quantitative parameters with tumor invasion and Ki-67 expression in early lung adenocarcinoma.The study involved 141 lesions in 141 patients with early lung adenocarcinoma. According to the degree of tumor invasion, the lesions were assigned into (adenocarcinoma in situ + minimally invasive adenocarcinoma) group and invasive adenocarcinoma (IAC) group. Artificial intelligence-assisted diagnostic software was used to automatically outline the lesions and extract corresponding quantitative parameters on CT images. Statistical analysis was performed to explore the correlation of these parameters with tumor invasion and Ki-67 expression.The results of logistic regression analysis showed that the short diameter of the lesion and the average CT value were independent predictors of IAC. Receiver operating characteristic curve analysis identified the average CT value as an independent predictor of IAC with the best performance, with the area under the receiver operating characteristic curve of 0.893 (P < .001), and the threshold of –450 HU. Besides, the predicted probability of logistic regression analysis model was detected to have the area under the curve of 0.931 (P < .001). The results of Spearman correlation analysis showed that the expression level of Ki-67 had the highest correlation with the average CT value of the lesion (r = 0.403, P < .001).The short diameter of the lesion and the average CT value are independent predictors of IAC, and the average CT value is significantly positively correlated with the expression of tumor Ki-67.

The Expression and Effect of ABCE1 in Gastric Adenocarcinoma.

ATP-binding cassette E1 (ABCE1) is mainly related to the regulation of viral infection, cell multiplication, and anti-apoptosis. Previous reports confirmed the central role in the regulation of ABCE1 in liver and breast cancer; however, its potential role in gastric adenocarcinoma remains unclear. In our study, siRNA and plasmid were transfected to construct gastric cancer cell lines with low and overexpression of ABCE1, and Western blot, RT-qPCR, and immunohistochemical staining were used to detect ABCE1 expression levels in gastric cancer tissues and cell lines. The effects of ABCE1 on cell growth, metastasis, invasion, cell cycle, and drug resistance were investigated using CCK-8 test, wound healing assay, and clone formation experiment. Functional experiments indicated that si-ABCE1 decreased the proliferation, metastasis, and invasion of gastric adenocarcinoma. Meanwhile, si-ABCE1 has significantly promoted EMT process and enhanced the sensitivity of paclitaxel and cisplatin. In vivo experiments also confirmed that si-ABCE1 group had significantly smaller tumors, and immunohistochemical staining results showed the tumor growth in si-ABCE1 group was reduced obviously. In summary, we found ABCE1 is considered as a crucial role in the evolution of gastric adenocarcinoma and could be a viable therapeutic target for the disease.

Predictors of Invasiveness in Adenocarcinoma of Lung with Lepidic Growth Pattern.

Lung adenocarcinoma with lepidic growth pattern (LPA) is characterized by tumor cell proliferation along intact alveolar walls, and further classified as adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive lepidic predominant adenocarcinoma (iLPA). Accurate diagnosis of lepidic lesions is critical for appropriate prognostication and management as five-year survival in patients with iLPA is lower than in those with AIS and MIA. We aimed to evaluate the accuracy of CT-guided core needle lung biopsy classifying LPA lesions and identify clinical and radiologic predictors of invasive disease in biopsied lesions. Thirty-four cases of adenocarcinoma with non-invasive lepidic growth pattern on core biopsy pathology that subsequently were resected between 2011 and 2018 were identified. Invasive LPA vs. non-invasive LPA (AIS or MIA) was defined based on explant pathology. Histopathology of core biopsy and resected tumor specimens was compared for concordance, and clinical, radiologic and pathologic variables were analyzed to assess for correlation with invasive disease. The majority of explanted tumors (70.6%) revealed invasive disease. Asian race (p = 0.03), history of extrathoracic malignancy (p = 0.02) and absence of smoking history (p = 0.03) were associated with invasive disease. CT-measured tumor size was not associated with invasiveness (p = 0.15). CT appearance of density (p = 0.61), shape (p = 0.78), and margin (p = 0.24) did not demonstrate a significant difference between the two subgroups. Invasiveness of tumors with lepidic growth patterns can be underestimated on transthoracic core needle biopsies. Asian race, absence of smoking, and history of extrathoracic malignancy were associated with invasive disease.

β2-adrenergic receptor drives the metastasis and invasion of pancreatic ductal adenocarcinoma through activating Cdc42 signaling pathway

Recent investigations indicate that β2-adrenergic receptor (β2-AR) signaling may facilitate the progression of various tumors, whose underlying mechanisms remain largely elusive. In the present study, we showed that β2-AR recruited Cdc42 in response to isoproterenol (ISO, a β-AR selective agonist) exposure in pancreatic ductal adenocarcinoma (PDAC) cells. The association of β2-AR and Cdc42 promoted the activation of Cdc42, as revealed by increased levels of Cdc42-GTP, and co-incubation with β2-AR antagonist abrogated ISO-induced activation of Cdc42. β2-AR-mediated Cdc42 activation further led to the phosphorylation of downstream PAK1, LIMK1 and Merlin. Furthermore, we showed that the activation of β2-AR/Cdc42 signaling facilitated the migration and invasion of PDAC cells. In addition, β2-AR and Cdc42 were overexpressed in PDAC specimens, compared with adjacent non-tumor tissues. High expression of β2-AR and Cdc42 were correlated with lymph node metastasis and TNM stage in PDAC patients. Finally, we showed that overexpression of β2-AR and Cdc42 were indicative of unfavorable prognosis in PDAC patients. Taken together, our findings suggested that β2-AR might facilitate Cdc42 signaling to drive the migration and invasion of PDAC cells, consequently resulting in the metastasis and dismal prognosis of PDAC. These studies highlight targeting β2-AR/Cdc42 signaling as a therapeutic strategy against PDAC.

Abstract 3463: Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to all treatment modalities. Early metastatic dissemination, rich desmoplastic stroma and immunosuppressive infiltrate are some of the features contributing to the poor prognosis of PDAC patients (5-year survival rate of less than 10%). Despite scientific progress in the development of new therapeutic regimes, PDAC patients in low- and middle-income countries have restricted access to high-cost innovative drugs. Recently, attention has been given to the concept of drug repurposing that could meet the need for novel but affordable and widely accessible therapies. Imidazoline receptor 1 Nischarin (NISCH, IR1, IRAS) is an integrin α5-interacting protein that acts as a tumor suppressor in breast cancer through modulation of cancer cell motility and survival. Importantly, NISCH has several clinically approved agonists that are used for the treatment of hypertension and are shown to modulate autophagy and reduce inflammation and fibrosis, which makes NISCH a potentially good therapeutic target for defying PDAC progression. Based on our GSEA analysis of NISCH expression in PDAC patient samples, NISCH was predicted to have an effect on cancer cell focal adhesion assembly and cytoskeletal organization. Therefore, we examined the effects of NISCH agonists on PDAC cells fitness in vitro. A panel of PDAC cell lines was tested in MTT assay to determine the effects of NISCH agonists rilmenidine, clonidine and moxonidine on cell viability. Flow cytometry, western blot and immunofluorescence were used to examine the effects of NISCH agonists on PDAC cell metabolism, migration and cytoskeletal organization. Tg(fli1:EGFP) zebrafish model was used to examine the effects of NISCH agonists on PDAC cell growth and invasion. We found that NISCH was expressed in PDAC cell lines, and that out of the three tested agonists, rilmenidine most potently inhibited cancer cell viability. Treatment with rilmenidine significantly reduced PANC-1, MIA PaCa-2 and BxPC-3 cell attachment to extracellular matrix (collagen type I and fibronectin) and migratory potential in wound healing and transwell assays. Furthermore, treatment with rilmenidine altered the organization of actin cytoskeleton and focal adhesion assembly, potentially through the reduction of intracellular reactive oxygen species (ROS) levels. Importantly, treatment with rilmenidine inhibited invasion of PANC-1 cells in Tg(fli1:EGFP) zebrafish model. With these antimigratory and potentially antimetastatic effects of rilmenidine, our study lays the ground for a more extensive examination of the biological role of NISCH in PDAC progression and implies that its agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Aleksandar Pavic, Tatjana Srdic-Rajic, Jelena Grahovac. Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3463.

Abstract 676: ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis

Abstract Lung cancer is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into invasive mesenchymal cells. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in a panel of lung adenocarcinoma cell lines. We hypothesize that ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis. We determined that the acetyl-deficient mutant (125kDa) exhibits a decreased half-life compared to WT and acetylated ZEB1, suggesting that disruption of acetylation hinders dimerization and protein stability. However, the acetyl-mimetic mutant (250kDa) protects ZEB1 from proteasomal degradation brought by the action of the E3 ubiquitin ligase SIAH1. siRNA-mediated silencing of SIAH1 increased the mRNA and protein levels of ZEB1 WT and both acetylation mutants. Importantly, silencing of SIAH1 markedly increased the half-life of 250kDa ZEB1 WT from 18.0hrs to 25.4hrs and acetyl-mimetic from 12.3hrs to 24.0hrs, as well as 125kDa ZEB1 acetyl-deficient from 9.7hrs to 18.3hrs. Although we determined that 250kDa ZEB1 is significantly more stable than 125kDa ZEB1, the increase in the stability by silencing SIAH1 contributes to ZEB1 acetyl-deficient binding at the promoter of its target genes mir200c-141 and SEMA3F. 250kDa ZEB1 WT and acetyl-mimetic recruits the histone deacetylases (HDACs) containing the nucleosome remodeling and deacetylase (NuRD) complex, to promote lung adenocarcinoma invasion and metastasis compared to ZEB1 acetyl-deficient. We further established that the stability of ZEB1 is integral to promoting these mesenchymal features. Our results suggest that ZEB1_K811ac regulates its dimerization and stability to promote lung adenocarcinoma via EMT. Future studies are focused on understanding the role of ZEB1 protein stability to recruit NuRD co-repressor and subsequently mediate tumorigenesis and metastasis. Citation Format: Mabel G. Perez-Oquendo, Roxsan Manshouri, Don Gibbons. ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 676.

Villin expression in human tumours: a tissue microarray study on 14,398 tumours

Background Villin is a protein of the brush border of epithelial cells which is used as an immunohistochemical marker for colorectal and gastrointestinal neoplasms. However, other tumor entities can also express villin. Methods To comprehensively determine villin expression, tissue microarrays containing 14,398 samples from 118 different tumor types as well as 608 samples of 76 different normal tissues were analyzed by immunohistochemistry. Results Villin was found in 54 of 118 tumor categories, including 36 tumor categories with strong staining. Villin expression was frequent in colorectal, upper gastrointestinal tract, pancreatobiliary, and renal tumors as well as in mucinous ovarian cancers, yolk sac tumors and in neuroendocrine neoplasms. Reduced villin expression was linked to advanced pT stage, lymph vessel invasion and microsatellite instability (p≤0.0006) in colorectal adenocarcinoma. In summary, our data demonstrate that villin expression is most common in gastrointestinal, pancreatobiliary, and neuroendocrine neoplasms, yolk sac tumors and mucinous ovarian cancers. Conclusion Our data support a high utility of villin immunohistochemistry for the identification of tumors with gastrointestinal, pancreatobiliary, and yolk sac tumor origin. However, considering that at least a weak villin positivity in some tumor cells occurred in 54 different tumor categories, villin immunohistochemistry should be applied as a part of a marker panel rather than as a stand-alone marker.

The Invasion and Metastasis of Colon Adenocarcinoma (COAD) Induced by SALL4

Objective The development and progression of many cancers may be related to SALL4, the role and molecular mechanism of which are unclear in colon adenocarcinoma (COAD). Methods The SALL4 expression in adjacent normal mucosa tissues and carcinoma tissues of patients with COAD was detected through bioinformatic analysis based on TCGA database and immunohistochemistry. Single-cell analysis showed that the expression of SALL4 in normal tissue was noticeably low. GSEA analysis suggested that the SALL4 upregulated the GO and pathway of growth and cancer development and downregulated metabolization pathway. The relationship between lymph node metastasis, histological grading, clinical staging, and the expression of SALL4 in carcinoma tissues was analyzed. The upregulated or downregulated SALL4 expression of COAD cell lines was established. The influence of SALL4 on COAD cells invasion and proliferation was detected using plate cloning assay and Transwell. The expressions of EMT-related proteins E-cadherin, N-cadherin, vimentin, and Twist were detected by Western blot. The EMT phenotype was analyzed by immunofluorescence. Results The study confirmed that the expression of SALL4 was upregulated in COAD and positively correlated with the degree of tumor differentiation, tumor staging, and metastasis. The overexpression of SALL4 was related to a poor prognosis, promoted the invasion and proliferation of colorectal cancer cells, and accelerated the occurrence of EMT, which was characterized by upregulation of Twist, vimentin, and N-cadherin expressions and downregulation of E-cadherin. The immunofluorescence staining confirmed the EMT phenotype. On the contrary, knocking out SALL4 gene reversed EMT, weakened cell proliferation and invasion, inhibited upregulation of Twist, vimentin, and N-cadherin expressions and downregulation of E-cadherin. Conclusion To sum up, TNM grading, histological grading, and lymphatic metastasis were significantly correlated with SALL4 in tumor tissues. SALL4 played a vital role in tumor proliferation, invasion, and tumor EMT and may be a novel target for COAD.

Cystatin SN promotes epithelial-mesenchymal transition and serves as a prognostic biomarker in lung adenocarcinoma

BackgroundCystatins are a class of proteins that can inhibit cysteine protease and are widely distributed in human bodily fluids and secretions. Cystatin SN (CST1), a member of the CST superfamily, is abnormally expressed in a variety of tumors. However, its effect on the occurrence and development of lung adenocarcinoma (LUAD) remains unclear.MethodsWe obtained transcriptome analysis data of CST1 from The Cancer Genome Atlas (TCGA) and GSE31210 databases. The association of CST1 expression with prognosis, gene mutations and tumor immune microenvironment was analyzed using public databases. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to investigate the potential mechanisms of CST1.ResultsIn this study, we found that CST1 was highly expressed in lung adenocarcinoma and was associated with prognosis and tumor immune microenvironment. Genetic mutations of CST1 were shown to be related to disease-free survival (DFS) by using the c-BioPortal tool. Potential proteins binding to CST1 were identified by constructing a protein-protein interaction (PPI) network. Gene set enrichment analysis (GSEA) of CST1 revealed that CST1 was notably enriched in epithelial-mesenchymal transition (EMT). Cell experiments confirmed that overexpression of CST1 promoted lung adenocarcinoma cells migration and invasion, while knockdown of CST1 significantly inhibited lung adenocarcinoma cells migration and invasion.ConclusionsOur comprehensive bioinformatics analyses revealed that CST1 may be a novel prognostic biomarker in LUAD. Experiments confirmed that CST1 promotes epithelial-mesenchymal transition in LUAD cells. These findings will help to better understand the distinct role of CST1 in LUAD.