Paxillin Research Articles

Trimethylamine N-Oxide Aggravates Neuro-Inflammation via lncRNA Fendrr/miR-145-5p/PXN Axis in Vascular Dementia Rats.

Vascular dementia (VaD) is the second most common dementia in the world. An increasing number of studies have demonstrated the important role of long non-coding RNAs (lncRNAs) in VaD. Our previous investigation demonstrated that Trimethylamine-N-oxide (TMAO) exacerbates cognitive impairment and neuropathological alterations in VaD rats. Thus, we hypothesized that TMAO could play an injury role in VaD by regulating lncRNAs. The rats using the bilateral common carotid artery (2VO) model were administered TMAO (120 mg/kg) for 8 consecutive weeks, 4weeks preoperatively and 4weeks postoperatively. High-throughput sequencing was conducted to investigate the effects of TMAO treatment on lncRNA expression in rat hippocampus and bioinformatics analysis was performed to identify potential downstream targets. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA fetal-lethal noncoding developmental regulatory RNA (Fendrr), miR-145-5p, and paxillin (PXN). Learning and spatial memory capacities were measured, as well as inflammatory factors. Nissl staining was used to observe neuronal injury in the CA1 area of the hippocampus. Furthermore, we used the Fendrr loss-of-function assay, miR-145-5p gain-of-function assays and PXN loss-of-function assay to explore the mechanisms by which TMAO acts on VaD. TMAO administration upregulated lncRNA Fendrr expression in the rat hippocampus, while the damaging effects of TMAO were counteracted after knockdown of Fendrr. Fendrr exhibits highly expressed in 2VO rats and sponged miR-145-5p, which targets PXN. Silencing of Fendrr or PXN, or promotion of miR-145-5p improved neurological function injury, reduced neuronal damage, as well as repressed inflammation response. Inhibition of miR-145-5p abrogated up Fendrr knockdown mediated influence on 2VO rats. The results of this study indicated that TMAO inhibits the miR-145-5p/PXN axis by increasing the Fendrr expression, thus exacerbating the development of VaD.

Novel biomarkers associated with oxidative stress and immune infiltration in intervertebral disc degeneration based on bioinformatics approaches

BackgroundThe etiology of intervertebral disc degeneration (IVDD), a prevalent degenerative disease in the elderly, remains to be fully elucidated. The objective of this study was to identify immune infiltration and oxidative stress (OS) biomarkers in IVDD, aiming to provide further insights into the intricate pathogenesis of IVDD. MethodsThe Gene Expression microarrays were obtained from the Gene Expression Omnibus (GEO) database. We conducted enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. Subsequently, the R language packages CIBERSORT, MCPcounter, and WGCNA were employed to compare immune infiltration levels between IVDD samples and control samples. A protein-protein interaction (PPI) network was constructed using the Search Tools for the Retrieval of Interacting Genes (STRING) database to identify significant gene clusters. To identify hub genes, we employed Cytoscape's Molecular Complex Detection (MCODE) plug-in. The mRNA levels of hub genes in the cell model were validated by qPCR, while Western blotting was used to validate their protein levels. ResultsThe GSE70362 dataset from the GEO database identified a total of 1799 genes that were differentially expressed. Among these, 43 genes were found to be differentially expressed and also associated with OS. The differentially expressed genes associated with OS and the immune-related module genes identified through WGCNA were further intersected, resulting in the identification of 10 key genes that were differentially expressed and played crucial roles in both immune response and OS. Subsequently, we validated four diagnostic markers (PPIA, MAP3K5, PXN, and JAK2) using the GSE122429 external dataset. In a cellular model of OS in NP cells, we have identified the upregulation of PPIA and PXN genes, which could serve as novel markers for IVDD. ConclusionThe study successfully identified and validated differentially expressed genes associated with oxidative stress and immune infiltration in IVDD samples compared to normal ones. Notably, the newly discovered biomarkers PPIA and PXN have not been previously reported in IVDD-related research.

Evaluation of Paxillin Expression in Odontogenic Cysts and Tumors.

Paxillin (PXN) is one of the proteins involved in cell adhesion. PXN and integrins constitute a key site for the focal adhesion between the cell and extracellular matrix. Several studies have shown that PXN is a factor in tumor formation, progression, invasion, and metastasis. This study evaluated PXN expression in four types of odontogenic lesions with different aggressive behaviors. In this retrospective cross-sectional study, PXN expression was immunohistochemically assessed in 68 paraffin-embedded tissue samples from patients with the confirmed diagnosis of four types of odontogenic lesions, including 14 dentigerous cysts (DC), 20 odontogenic keratocyst (OKC), 16 unicystic ameloblastoma, and 18 solid ameloblastoma. The PXN expression in these samples were scored based on the percentage and intensity of immunoreactivity, and compared among the groups by Chi-square test. The PXN marker was detected in the cytoplasm of tumor cells (unicystic and solid ameloblastoma) and the epithelial layer of cysts (DC and OKC). The intensively stained marker of PXN was observed in 9 cases (64.3%) of the DC, 14 cases (70%) of OKC, 12 cases (75%) of unicystic ameloblastoma, and 13 cases (72.2%) of solid ameloblastoma. However, there was not statistical difference of PXN protein expression between DC and OKC (p Value = 0.51) and unicystic and solid ameloblastoma (p = 0.58), also the same was true for cysts and tumors (p = 0.37). The expression of PXN is not related to the biological behaviors of odontogenic lesions.

Abstract 5418: Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells

Abstract Hypoxia is a physiologically relevant feature of the prostate microenvironment that promotes migration and invasion resulting in extracapsular extension, the first step toward metastatic progression. Prostate cancer invasion depends on integrins as mechanosensing membrane receptors during creation of membrane lamellipodial protrusions and focal adhesions (FAs). The objective of this study was to determine the molecular events that promote membrane protrusions under hypoxia and whether this was dependent upon kindlin-2, an essential integrin adapter that marks activated β1 integrin heterodimers. DU145 cells depleted of one copy of FERMT2+/- (50% kindlin-2 expression) by CRISPR/Cas9 or DU145 FERMT2wt (normal kindlin-2 expression) were grown under acute exposure to hypoxia (1% O2) and compared to cells grown under normal tissue culture conditions. Immunofluorescence microscopy experiments were performed to analyze the spatial temporal expression of kindlin-2 complexes. Kindlin-2 complexes were confirmed by immunoprecipitation using an anti-kindlin-2 3A3-antibody from Sigma-Aldrich. Colocalization was determined by obtaining 2D immunofluorescence microscopy images analyzed using ImageJ 2.1.0/1.53c and Nikon NIS-Elements 5.30.04. Under hypoxic conditions, analysis over four time points (4h, 8h, 12h & 16h) increased the number and area of FAs (marked by paxillin (PXN)) containing kindlin-2 in a time-dependent manner by 2-fold and 1.5-fold, respectively, but not in DU145 FERMT2+/- cells. Additionally, hypoxia increased membrane area, perimeter, and the plasma membrane intensity of kindlin-2 exclusive of FAs in DU145 FERMT2wt cells which was maximal after 8 hours of hypoxia. Interestingly, limiting the kindlin-2 expression in FERMT2+/- cells resulted in a loss of hypoxia-induced lamellipodial protrusions (marked by lamellipodin (RAPH1)) containing kindlin-2 while hypoxia-induced kindlin-2 FA changes were preserved. The current working hypothesis is that lamellipodial protrusions are dependent upon kindlin-2 expression whereas the established FAs are stable under conditions of reduced kindlin-2 expression in hypoxia. This data suggests that an early stage of migration, lamellipodial extensions are sensitive to kindlin-2 availability. Further studies are required to determine the dynamic interplay between protrusive events and focal adhesions in relation to kindlin-2 for prostate cancer cells to migrate and invade. Citation Format: Colin Nelson, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec, Anne E. Cress. Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells [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 5418.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-β estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Effect of Paxillin Expression and Phosphorylation on Colorectal Cancer Prognosis and Metastasis.

Colorectal cancer (CRC) is the third most common cancer worldwide, and metastasis is strongly associated with poor prognosis in patients with CRC. We have previously found that the expression and phosphorylation of paxillin (PXN) play an important role in the metastatic potential of breast cancer. This study examined the potential role of PXN in CRC metastasis. Resected tumor specimens from 92 patients with CRC were subjected to immunohistochemical analysis of PXN levels. Three human CRC cell lines, HCT116, LoVo, and SW480 were used for scratch and transwell invasion assays to examine the effects of PXN over-expression. RNA sequencing was performed to obtain the expression profiles under PXN over-expression. High levels of PXN were significantly correlated with advanced stage, higher carcinoembryonic antigen and carbohydrate antigen 19-9 levels, and poorer overall survival. The migration ability of CRC cells was enhanced by exogenous PXN over-expression, but this enhancement was not observed in cells harboring exogenously mutated PXN at Tyr31 or Tyr88 phosphorylation sites. In PXN-over-expressing cells, TNF-α signaling via NF-[Formula: see text]B was positively enriched. PXN expression and phosphorylation at Tyr31 or Tyr88 may influence the migration and invasion of CRC cells. PXN expression and phosphorylation at Tyr31 or Tyr88 are promising targets for evaluating prognosis and treating CRC.

265 Interactive Effects of Dietary Isoleucine and Valine Ratios to Lysine in Response to Varying Leucine to Lysine Ratios on Pectoralis Major Muscle Protein Expression in Commercial Broilers

Abstract Optimizing the dietary concentrations and ratios of branched-chain amino acids (BCAA) is necessary to optimize broiler chicken growth performance and carcass meat yield. A central composite design (CCD) study was conducted to understand the impact of dietary BCAA concentrations and ratios on Pectoralis major (PM) muscle protein expression involved in mTOR and related protein synthesis pathways as well as and investigate the mechanisms behind how BCAA ratios affect broiler growth and muscle protein accretion. A total of 2,592 d-old Ross 344 × 708 male broilers were randomly placed in 144 floor pens (n = 18 birds per pen). Before the initiation of the experimental finisher phase, all pens were standardized to contain 15 birds per pen. Each pen received 1 of 15 dietary treatments in the 23 CCD with 6 center points from 20 to 35 d of age, varying in digestible ratios of valine:lysine (Val:Lys; 64 to 87), leucine:lysine (Leu:Lys; 110 to 185), and isoleucine:lysine (Ile:Lys; 52 to 75). On d 35, 1 bird per pen was randomly selected for PM protein extraction and proteomic analysis via data independent acquisition protein sequencing with a timsTOF Pro 2 LC/MS/MS and Spectronaut 15 software. Ribosomal protein S6 kinase (RPS6KA3), eukaryotic elongation factor 2 (EEF2), eukaryotic translation initiation factor 4B (EIF4B), eukaryotic translation initiation factor 4E (EIF4E), CAP-Glycine domain containing linker protein 1 (CLIP1), mitogen-activated protein kinase 3 (MAPK3), Protein kinase domain-containing protein (MAP2K1), Dual-specificity mitogen-activated protein kinase kinase 2 (MAP2K2), paxillin (PXN), and regulator complex protein (LAMTOR3) were identified, quantified, and analyzed as a CCD using the RSREG procedure of SAS ver. 9.4 with significance set at P ≤ 0.10. The surface response models for RPS6KA3, EEF2, EIF4B, EIF4E, MAPK3, MAP2K1, MAP2K2, CLIP1, PXN, and LAMTOR3 were not significant (P ≥ 0.926; R2 values ≤ 0.1). No linear (P ≥ 0.5223), quadratic (P ≥ 0.0.634) or cross product (P ≥ 0.6238) regression effects were observed for any of the proteins analyzed. A minimum stationary point was observed for RPS6KA3, EEF2, MAPK3, and LAMTOR3. However, optimal values for the other proteins could not be obtained due to stationary saddle points. Overall, the results in this study indicate that varying concentrations of dietary BCAA may not impact expression of proteins related to broiler skeletal muscle growth on d 35 following the 15-d treatment period, despite the influence on live performance. This is a limitation of this study and suggests that if the mTOR pathway was involved it was not still differentially up- or down-regulated at this time point. Further investigation and new studies with multiple time points will be required to better understand the mechanisms behind how BCAA impact broiler growth and muscle development.

Angiopoietin-like 4 induces head and neck squamous cell carcinoma cell migration through the NRP1/ABL1/PXN pathway

ObjectivesThe molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell carcinoma (HNSCC) growth and dissemination are unclear. Materials and MethodsWe investigated ANGPTL4 expression in human normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), oral leukoplakia cells (LEUK1), and HNSCC cell lines, as well as in tissue biopsies from patients with oral dysplasia, and primary and metastatic HNSCC. We further examined the contribution of ANGPTL4 cancer progression in an HNSCC orthotopic floor-of mouth tumor model and the signaling pathways linking ANGPTL4 to cancer cell migration. ResultsANGPTL4 expression was upregulated in premalignant DOKs and HNSCC cell lines compared to NOKs and was increased in tissue biopsies from patients with oral dysplasia, as well as in primary and metastatic HNSCC. We also observed that downregulation of ANGPTL4 expression inhibited primary and metastatic cancer growth in an HNSCC orthotopic tumor model. Interestingly, ANGPTL4 binding to the neuropilin1 (NRP1) receptor led to phosphorylation of the focal adhesion protein, paxillin (PXN), and tumor cell migration; this was dependent on the tyrosine kinase ABL1. Treatment with the ABL1 inhibitor, dasatinib and small interfering RNA silencing of NRP1 or ABL1 expression blocked PXN phosphorylation and tumor cell migration. ConclusionOur findings suggest an early, sustained, and angiogenesis-independent autocrine role for ANGPTL4 in HNSCC progression and expose ANGPTL4/NRP1/ABL1/PXN as an early molecular marker and vulnerable target for the prevention of HNSCC growth and metastasis.

Paxillin is a potential prognostic biomarker associated with immune cell infiltration in ovarian cancer

ObjectiveTo investigate the expression, prognosis, and underlying mechanism of Paxillin (PXN) in ovarian cancer. Materials and methodsBy comprehensive use of various bioinformatics tools, we analyzed the expression of PXN and its prognostic value in ovarian cancer. Then, the enrichment analyses were conducted to determine the possible regulatory pathways PXN involved in ovarian cancer. Finally, the associations of PXN expression with immune cell infiltration and immune checkpoints were analyzed. ResultsPXN was highly expressed in ovarian cancer and its expression could independently predict the overall survival of ovarian cancer patients. More importantly, PXN had a superior ability in predicting long-term survival than age and tumor residual disease in ovarian cancer patients. In addition, PXN was positively related to adherens junction and tight junction pathways. Significant negative relationships between PXN expression and immune infiltrates were observed, however, PXN was positively connected with immune checkpoint (VSIR) in ovarian cancer. ConclusionsPXN serves as a reliable prognostic biomarker and may be a potent therapeutic target for ovarian cancer. Moreover, high PXN expression may affect ovarian cancer progression via positive regulation of metastasis-related pathways.

Pan-Cancer Analysis on the Oncogenic Role of Programmed Cell Death 10.

Purpose Programmed cell death factor 10 (PDCD10) is associated with intercellular junction, cytoskeleton organization, cell proliferation, apoptosis, exocytosis, and angiogenesis. However, the role of PDCD10 in human cancer is unclear. This study aims to explore the role of PDCD10 in various tumors and its possible mechanism through bioinformatics analysis. Methods We verified the expression of the PDCD10 gene based on data from the ONCOMINE, TIMER2.0, and TISDB databases. The correlation of PDCD10 with prognosis of patients with different tumors was analyzed using data from the GEPIA2 database. Proteins bound to PDCD10 were analyzed from the STRING database. PDCD10, PDCD10-binding proteins, and associated candidate genes were analyzed in DAVID for functional and pathway analyses. We also evaluated the immunological, clinical, and genetic aspects of distinct cancers by using TIMER2.0 and the connection between PDCD10 expression and tumor immune subtypes by using TISDB. Single-cell sequencing data from the CancerSEA database were used to characterize cancer cell functional states and generate heat maps. Results PDCD10 overexpression is linked to certain molecular subtypes of human cancer. Low PDCD10 expression in patients with bladder urothelial carcinoma (BLCA), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), adrenocortical carcinoma (ACC), head and neck squamous cell carcinoma (HNSC), kidney chromophobe carcinoma (KICH), brain lower grade glioma (LGG), pancreatic adenocarcinoma (PAAD), uterine corpus endometrial carcinoma (UCEC), oral squamous cell carcinoma (OSCC), and esophageal adenocarcinoma (ESAD) was correlated with favorable OS, whereas high PDCD10 expression in patients with LUSC, KIRC, READ, SKCM, and THYM was correlated with good prognosis. STRING network prediction results showed that 20 proteins, namely, paxillin (PXN), CCM2 scaffold protein (CCM2), TRAF3 interacting protein 3 (TRAF3IP3), FGFR1 oncogene partner 2 (FGFR1OP2), chromosome 4 open reading frame 19 (C4orf19), suppressor of IKBKE 1 (SIKE1), serine/threonine kinase 25 (STK25), striatin (STRN), protein phosphatase 2 catalytic subunit alpha (PPP2CA), mammalian sterile-20-like kinase 4 (MST4), MOB family member 4 (MOB4), protein phosphatase 2 scaffold subunit Abeta (PPP2R1B), sarcolemma-associated protein (SLMAP), serine/threonine kinase 24 (STK24), striatin 4 (STRN4), STRN3, protein phosphatase 2 scaffold subunit A alpha (PPP2R1A), striatin interacting protein 1 (STRIP1), CTTNBP2 N-terminal like (CTTNBP2NL), and cortactin binding protein 2 (CTTNBP2), can bind to PDCD10. Gene enrichment analysis suggested that PDCD10 is involved in the occurrence of different tumors through the Hippo signalling pathway, RNA transport, mRNA monitoring pathway, endocytosis, and T cell receptor signalling pathway. An inverse relationship was found between PDCD10 expression and cancer-associated fibroblasts in LUSC and TGCT, and PDCD10 expression was strongly connected with immunological subtypes, such as C1 (wound healing), C2 (interferon-gamma dominant), C3 (inflammation), C4 (lymphocyte depletion), C5 (immune silenced), and C6 (TGF-beta dominant). Finally, analysis of single-cell sequencing data revealed that PDCD10 expression is linked to epigenetic reprogramming, DNA repair, cell cycle progression, cell differentiation, inflammation, cell proliferation, cell differentiation, cell invasion, and angiogenesis. Conclusion The results of our investigation demonstrate that PDCD10 has an oncogenic function in many cancer types. This study provides a reference for future research on antitumor therapeutic targets.

Identification of Paxillin as a Prognostic Factor for Glioblastoma via Integrated Bioinformatics Analysis.

Glioblastoma (GBM) is the most prevalent and aggressive type of brain tumor in the central nervous system. Clinical outcomes for patients with GBM are unsatisfactory. Here, we aimed to identify novel, reliable prognostic factors for GBM. Cox and interactive analyses were used to identify hub genes from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas datasets. After validation using various cohorts, survival analysis, meta-analysis, and prognostic analysis were performed. Coexpression and enrichment analyses were performed to elucidate the biological pathways of hub genes involved in GBM. ESTIMATE and CIBERSORT methods were applied to analyze the association of hub genes with the tumor microenvironment (TME). Paxillin (PXN) was identified as a hub gene with a high expression in GBM. PXN expression was negatively correlated with overall survival, progression-free survival, and disease-free survival in patients with GBM. Meta-analysis and Cox analysis revealed that PXN could act as an independent prognostic factor in GBM. In addition, PXN was significantly coexpressed with signal transducer and activator of transcription 3 and transforming growth factor β1 and participated in focal adhesion, extracellular matrix/receptor interactions, and the phosphatidylinositol 3-kinase/AKT signaling pathway. The results of ESTIMATE and CIBERSORT analyses revealed that PXN was implicated in TME alterations, particularly the infiltration of regulatory T cells, activated memory T cells, and activated natural killer cells. PXN may be a reliable prognostic factor for GBM. Further studies are needed to validate these findings.

Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [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 3835.

Repositioning of experimentally validated anti-breast cancer peptides to target FAK-PAX complex to halt the breast cancer progression: a biomolecular simulation approach.

FAK (focal adhesin kinase), a tyrosine kinase, plays an imperative role in cell-cell communication, particularly in cell signaling systems. It is a multi-functional signaling protein, which integrates and transduces signals into cancer cells through growth factor receptors or integrin and its interaction with Paxillin (PAX). The molecular processes by which FAK promotes the development and progression of cancer have progressively established the possible relationship between FAK-PAX complex in many types of cancer. The interaction of FAX and PAX is very important in breast cancer and thus acts as an essential biomarker for drugs, vaccines or peptide inhibitor designing. In this regard, computational approaches, particularly peptide designing to target the binding interface of the interacting partners, would greatly assist the design of peptide inhibitors against various cancer. Accordingly, in this present study, we screened 236 experimentally validated anti-breast cancer peptides using computational drugs repositioning approach to design peptides targeting the FAK-PAX complex. Using protein-peptide docking the binding site for the HP1 was confirmed and a total of 236 anti-breast cancer peptides were screened. Among the 236, only 12 peptides reported a docking score better than the control. From these 12, Magainin with the docking score - 103.8 ± 10.3kcal/mol, NRC-07 with the docking score - 100.8 ± 16.5kcal/mol, and Indolicidin with the docking score - 101.7 ± 3.9kcal/mol, peptides potentially inhibit the FAX-PAX binding. Calculation of protein's motion and FEL revealed the binding and inhibitory behavior. Moreover, binding free energy (MM/GBSA) confirmed that Magainin exhibited the total binding energy - 53.28kcal/mol, NRC-07 possessed the TBE - 44.16kcal/mol, and Indolicidin reported the TBE of - 40.48kcal/mol, thus explaining the inhibitory potential of these peptides. In conclusion, these peptides exhibit strong inhibitory potential and could abrogate the FAK-PAX complex in in vitro models and thus may relieve the burden of breast cancer.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin β1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

Identification of miR-199-5p and miR-199-3p Target Genes: Paxillin Facilities Cancer Cell Aggressiveness in Head and Neck Squamous Cell Carcinoma.

Our previous study revealed that the miR-199 family (miR-199a-5p/-3p and miR-199b-5p/-3p) acts as tumor-suppressive miRNAs in head and neck squamous cell carcinoma (HNSCC). Furthermore, recent studies have indicated that the passenger strands of miRNAs are involved in cancer pathogenesis. The aim of this study was to identify cancer-promoting genes commonly regulated by miR-199-5p and miR-199-3p in HNSCC cells. Our in silico analysis and luciferase reporter assay identified paxillin (PXN) as a direct target of both miR-199-5p and miR-199-3p in HNSCC cells. Analysis of the cancer genome atlas (TCGA) database showed that expression of PXN significantly predicted a worse prognosis (5-year overall survival rate; p = 0.0283). PXN expression was identified as an independent factor predicting patient survival according to multivariate Cox regression analyses (p = 0.0452). Overexpression of PXN was detected in HNSCC clinical specimens by immunostaining. Functional assays in HNSCC cells showed that knockdown of PXN expression attenuated cancer cell migration and invasion, suggesting that aberrant expression of PXN contributed to HNSCC cell aggressiveness. Our miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Exploring the Mechanism of the miRNA-145/Paxillin Axis in Cell Metabolism During VEGF-A-Induced Corneal Angiogenesis.

PurposePaxillin (PXN) is a key component of focal adhesions and plays an important role in angiogenesis. The aim of the present study was to investigate the effect of PXN in vascular endothelial growth factor A (VEGF-A)–induced angiogenesis in human umbilical vein endothelial cells (HUVECs).MethodsHUVECs were transfected with PXN overexpression and PXN interference vectors. Biochemical detection was used to detect adenosine triphosphate and lactic acid production. The morphology of mitochondria was observed under an electron microscope, and flow cytometry was conducted to measure mitochondrial membrane potential. Transwell experiments were used to detect the migration and tube formation ability of each group of cells. The expression of hexokinase (HK)1, HK2, glucose transporter 1 (GLUT1), phosphorylated phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, and phosphorylated mechanistic target of rapamycin (mTOR) was evaluated by western blot.ResultsPXN silencing reduced the levels of lactic acid and adenosine triphosphate, downregulated HK1, HK2, and GLUT1, suppressed PI3K/AKT/mTOR signaling activation, and inhibited VEGF-A–induced mitochondria injury in VEGF-A–induced HUVECs. We also determined that miR-145-5p decreased the VEGF-A–induced expression of PXN and inhibited the invasion and angiogenesis of HUVECs. Also, miR-145-5p inhibition blocked the protective effect of PXN interference on VEGF-A–induced HUVEC injury. Furthermore, PXN interference significantly decreased lactic acid and adenosine triphosphate levels, inhibited PI3K/AKT/mTOR activation, and decreased the levels of HK1, HK2, and GLUT1 in VEGF-A-treated mouse corneal.ConclusionsThe results indicate that PXN silencing inhibited the VEGF-A–induced invasion and angiogenesis of HUVECs via regulation of cell metabolism and mitochondrial damage, suggesting that PXN may be a potential target for antiangiogenic therapies.

MiR-497 defect contributes to gastric cancer tumorigenesis and progression via regulating CDC42/ITGB1/FAK/PXN/AKT signaling

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. MicroRNAs (miRNAs) are known to be important regulators of GC. This study aims to investigate the role of miRNA (miR)-497 in GC. We demonstrated that the expression of miR-497 was downregulated in human GC tissues. After N-methyl-N-nitrosourea treatment, the incidence of GC in miR-497 knockout mice was significantly higher than that in wild-type mice. miR-497 overexpression suppressed GC cell proliferation, cell-cycle progression, colony formation, anti-apoptosis ability, and cell migration and invasion capacity. Additionally, miR-497 overexpression decreased the expression levels of cell division cycle 42 (CDC42) and integrin β1 (ITGB1) and inhibited the phosphorylation of focal adhesion kinase (FAK), paxillin (PXN), and serine-threonine protein kinase (AKT). Furthermore, overexpression of miR-497 inhibited the metastasis of GC cells in vivo, which could be counteracted by CDC42 restoration. Furthermore, the focal adhesion of GC cells was found to be regulated by miR-497/CDC42 axis via ITGB1/FAK/PXN/AKT signaling. Collectively, it is concluded that miR-497 plays an important role in the repression of GC tumorigenesis and progression, partly via the CDC42/ITGB1/FAK/PXN/AKT pathway.

Pan-cancer analysis reveals an immunological role and prognostic potential of PXN in human cancer.

Paxillin (PXN) is a protein involved in numerous physiological processes, and its presence is closely related to the occurrence and development of many types of tumors. However, no studies have analyzed PXN from a pan-cancer perspective. We analyzed PXN expression, immune cell infiltration, prognosis, and biological function across different types of tumors included in The Cancer Genome Atlas and Gene Expression Omnibus datasets. The results showed that expression of PXN varies in different tumors. Expression of PXN strongly correlated with prognosis in patients with tumors; higher PXN expression usually was linked to poor overall and disease-free survival. Expression of PXN in breast invasive carcinoma and lymphoid neoplasm diffuse large B-cell lymphoma was related to the degree of CD8+ T-cell infiltration, and infiltration of cancer-associated fibroblasts, such as kidney renal papillary cell carcinoma and brain lower-grade glioma, was also observed in other tumors. The results of pan-cancer analysis showed that abnormal PXN expression was related to poor prognosis, immune infiltration, and protein phosphorylation in different tumor types. Therefore, the PXN gene may become a potential biomarker of clinical tumor prognosis.

CTCF-silenced miR-137 contributes to EMT and radioresistance in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in gastrointestinal system. MicroRNAs (miRNAs) have been reported to be implicated in cancer development. However, the role of miR-137 has not been fully revealed in ESCC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses were separately used to examine RNA level and protein level. 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assays and flow cytometry analyses were conducted to assess biological behaviors of ESCC cells. Additionally, the interaction between genes were analyzed via Chromatin Immunoprecipitation (ChIP) assay, RNA Binding Protein Immunoprecipitation (RIP) assay, RNA pull down assay and luciferase reporter assay.ResultsMiR-137 was down-regulated in ESCC cells. Upregulation of miR-137 hindered ESCC cell proliferation, migration, invasion and epithelial mesenchymal transition (EMT). Besides, miR-137 enhanced the sensitivity of ESCC cells to irradiation. Moreover, CCCTC-binding factor (CTCF) inactivated miR-137 transcription in ESCC cells. Furthermore, we revealed enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and paxillin (PXN) as the downstream targets of miR-137. In turn, EZH2 was recruited by CTCF and induced methylation in miR-137 promoter.ConclusionCTCF/Suz12/EZH2 complex-silenced miR-137 facilitates ESCC progression and radioresistance by targeting EZH2 and PXN.

Abnormal level of paxillin in cervical cancer cells is involved in tumor progression and invasion

Human papillomavirus (HPV) is the primary causative agent for the uterine cervical cancer. The expression of oncoproteins E6/E7 promotes apoptosis inhibition and increases the risk of cervical cancer progression. Some research reported that elevated expression of paxillin (PXN) stimulated cancer growth and invasion. However, the clinical significance of PXN in cervical cancer has not been well characterized so far. We found that PXN mRNA expression and protein level are significantly upregulated in cervical cancer cells compared to adjacent normal cells. Furthermore, the paxillin over-expression was correlated with potential of tumorigenesis and invasion. Cervical cancer cells with increased paxillin expression had an ability to form more tumor clones and were characterized by higher invasiveness as well. Therefore, our findings suggest that paxillin may act as an important prognostic factor for cervical cancer patients as it promotes tumor regeneration and invasion.