Mesenchymal Transition Markers Research Articles

Pan-cancer analyses suggest kindlin-associated global mechanochemical alterations

Kindlins serve as mechanosensitive adapters, transducing extracellular mechanical cues to intracellular biochemical signals and thus, their perturbations potentially lead to cancer progressions. Despite the kindlin involvement in tumor development, understanding their genetic and mechanochemical characteristics across different cancers remains elusive. Here, we thoroughly examined genetic alterations in kindlins across more than 10,000 patients with 33 cancer types. Our findings reveal cancer-specific alterations, particularly prevalent in advanced tumor stage and during metastatic onset. We observed a significant co-alteration between kindlins and mechanochemical proteome in various tumors through the activation of cancer-related pathways and adverse survival outcomes. Leveraging normal mode analysis, we predicted structural consequences of cancer-specific kindlin mutations, highlighting potential impacts on stability and downstream signaling pathways. Our study unraveled alterations in epithelial–mesenchymal transition markers associated with kindlin activity. This comprehensive analysis provides a resource for guiding future mechanistic investigations and therapeutic strategies targeting the roles of kindlins in cancer treatment.

Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [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 5445.

Identification of a novel gene signature related to prognosis and metastasis in gastric cancer.

Gastric Cancer (GC) presents poor outcome, which is consequence of the high incidence of recurrence and metastasis at early stages. GC patients presenting recurrent or metastatic disease display a median life expectancy of only 8 months. The mechanisms underlying GC progression remain poorly understood. We took advantage of public available GC datasets from TCGA using GEPIA, and identified the matched genes among the 100 genes most significantly associated with overall survival (OS) and disease free survival (DFS). Results were confirmed in ACRG cohort and in over 2000 GC cases obtained from several cohorts integrated using our own analysis pipeline. The Kaplan-Meier method and multivariate Cox regression analyses were used for prognostic significance and linear modelling and correlation analyses for association with clinic-pathological parameters and biological hallmarks. In vitro and in vivo functional studies were performed in GC cells with candidate genes and the related molecular pathways were studied by RNA sequencing. High expression of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 individually and as a signature was associated with poor prognosis and recurrent disease in GC. Moreover, the expression of ANKRD6 and ITIH3 was significantly higher in metastasis and their levels associated to Epithelial to Mesenchymal Transition (EMT) and stemness markers. In line with this, RNAseq analysis revealed genes involved in EMT differentially expressed in ANKRD6 silencing cells. Finally, ANKRD6 silencing in GC metastatic cells showed impairment in GC tumorigenic and metastatic traits in vitro and in vivo. Our study identified a novel signature involved in GC malignancy and prognosis, and revealed a novel pro-metastatic role of ANKRD6 in GC.

SLC26A9 promotes colorectal tumorigenesis by modulating Wnt/β-catenin signaling

Solute carrier family 26 member 9 (SLC26A9) is a member of the Slc26a family of multifunctional anion transporters that functions as a Cl- channel in parietal cells during acid secretion. We explored the role of SLC26A9 in colorectal cancer (CRC) and its related mechanisms through clinical samples from CRC patients, CRC cell lines and mouse models. We observed that SLC26A9 was expressed at low levels in the cytoplasm of adjacent tissues, polyps and adenomas but was significantly increased in colorectal adenocarcinoma. Moreover, increased levels of SLC26A9 were associated with a high risk of disease and poor prognosis. In addition, downregulation of SLC26A9 in CRC cells induced cell cycle arrest and apoptosis but inhibited cell proliferation and xenograft tumor growth both in vitro and in vivo. Mechanistic analysis revealed that SLC26A9 was colocalized with β-catenin in the nucleus of CRC cells. The translocation of these two proteins from the cytoplasm to the nucleus reflected the activation of Wnt/β-catenin signaling, and promoted the transcription of downstream target proteins, including CyclinD1, c-Myc and Snail, but inhibited the expression of cytochrome C (Cyt-c), cleaved Caspase9, cleaved Caspase3 and apoptosis-inducing factor (AIF). CRC is accompanied by alteration of epithelial mesenchymal transition (EMT) markers. Meanwhile, further studies showed that in SW48 cells, overexpressing SLC26A9 was cocultured with the β-catenin inhibitor XAV-939, β-catenin was downregulated, and EMT was reversed. Our study demonstrated SLC26A9 may be responsible for alterations in the proliferative ability and aggressive potential of CRC by regulating the Wnt/β-catenin signaling pathway.

Novel prognostic marker TGFBI affects the migration and invasion function of ovarian cancer cells and activates the integrin αvβ3-PI3K-Akt signaling pathway.

Individual patients with ovarian cancer show remarkably different prognosis. Present prognostic models for ovarian cancer mainly focus on clinico-pathological parameters, so quantifiable prognostic markers at molecular level are urgently needed. Platelets contribute to ovarian cancer progression, but have not been considered as biomarkers likely due to their instability. Here, we aimed to search for a stable prognostic marker from platelet-treated ovarian cancer cells, and explore its functions and mechanisms. Microarrays analysis was done with platelet-treated SKOV-3 ovarian cancer cells. Relevant studies were searched in the Gene Expression Omnibus (GEO) database. The candidate genes were determined by differentially expressed genes (DEGs), Venn diagram drawing, protein-protein interaction (PPI) network, Cox proportional hazards model and Kaplan-Meier analysis. The expression of TGFBI in clinical samples was assessed by immunehistochemical staining (IHC), and the association of TGFBI levels with the clinic-pathological characteristics and prognosis in ovarian cancer patients was evaluated by univariate and multivariate analysis. The functions of TGFBI were predicted using data from TCGA, and validated by in vitro and in vivo experiments. The mechanism exploration was performed based on proteomic analysis, molecular docking and intervention study. TGFBI was significantly higher expressed in the platelet-treated ovarian cancer cells. An analysis of bioinformatics data revealed that increased expression of TGFBI led to significant decrease of overall survival (OS), progression-free survival (PFS) and post-progression survival (PPS) in ovarian cancer patients. Tissue microarray results showed that TGFBI was an independent factor for ovarian cancer, and TGFBI expression predict poor prognosis. Functionally, TGFBI affected the migration and invasion of ovarian cancer cells by regulation of epithelial mesenchymal transition (EMT) markers (CDH1 and CDH2) and extracellular matrix (ECM) degradation proteins (MMP-2). Mechanistically, TGFBI phosphorylated PI3K and Akt by combining integrin αvβ3. We found out TGFBI as a novel prognostic indicator for ovarian cancer patients. TGFBI could promote metastasis in ovarian cancer by EMT induction and ECM remodeling, which might be associated with the activation of integrin αvβ3-PI3K-Akt signaling pathway.

Exposure of piperlongumine attenuates stemness and epithelial to mesenchymal transition phenotype with more potent anti-metastatic activity in SOX9 deficient human lung cancer cells.

Phytocompounds have shown hopeful results in cancer therapy. Piperlongumine (PIP), a naturally derived bioactive alkaloid found in our dietary spice, exhibits promising pharmacological relevance including anticancer activity. This study reconnoitred the anti-lung cancer effect of PIP and the allied mechanisms, in vitro and ex vivo. The cytotoxic, anti-proliferative, and apoptotic effects of PIP on lung cancer cells (LCC) were checked via cell viability, colony formation, cell migration, invasion, comet assay, and various staining techniques. Further, multicellular spheroids assay explored the anti-lung cancer potential of PIP, ex vivo. Preliminary results explored that PIP exerts selective cytotoxic and anti-proliferative effects on LCC by DNA damage and cell cycle arrest. PIP remarkably escalated the cellular and mitochondrial reactive oxygen species (ROS) generation and promoted dissipation of mitochondrial membrane potential (MMP), which triggers activation of caspase-dependent apoptotic pathway in LCC. Mechanistically, PIP showed F-actin deformation mediated significant anti-migratory and anti-invasive activity against LCC. Herein, we also found that F-actin dis-organization modulates the expression of epithelial to mesenchymal transition (EMT) markers and inhibits the expression of stemness marker proteins, like SOX9, CD-133, and CD-44. Moreover, PIP effectively reduced the size of spheroids with strong apoptotic and cytotoxic effects, ex vivo. This has been the first study to discover the high expression of SOX9 supporting the survival of LCC, whereas its inhibition induces higher sensitivity to PIP treatment. This study concludes a newer therapeutic agent (PIP) with promising anticancer activity against LCC by escalating ROS and attenuating MMP, stemness, and EMT.

Epithelial – mesenchymal transition markers, proliferation markers, and cytokine secretion in breast tissue in malignant and benign breast diseases

Epithelial – mesenchymal transition markers, proliferation markers, and cytokine secretion in breast tissue in malignant and benign breast diseases

Inhibition of proteinase-activated receptor 2 (PAR2) decreased the malignant progression of lung cancer cells and increased the sensitivity to chemotherapy

ObjectivesThis study aimed to study the effect of protease-activated receptor 2 (PAR2) on the proliferation, invasion, and clone formation of lung cancer cells. It also aimed to evaluate the inhibitory effect of melittin on PAR2 and the anti-lung cancer effect of melittin combined with gefitinib.MethodsThe correlation between the co-expression of PAR2 and epithelial–mesenchymal transition (EMT) markers was analyzed. PAR2 in A549 and NCI-H1299 cells was knocked down using siRNA. MTT assay, Transwell assay, and colony formation assay were used to detect the effects of PAR2 on cell proliferation, invasion, and clone formation. The anti-cancer effect of PAR2 knockdown on gefitinib treatment was analyzed. The synergistic effect of melittin on gefitinib treatment by inhibiting PAR2 and the underlying molecular mechanism were further analyzed and tested.ResultsThe expression of PAR2 was upregulated in lung cancer, which was associated with the poor prognosis of lung cancer. PAR2 knockdown inhibited the stemness and EMT of lung cancer cells. It also inhibited the proliferation, invasion, and colony formation of A549 and NCI-H1299 cells. Moreover, PAR2 knockdown increased the chemotherapeutic sensitivity of gefitinib in lung cancer. Melittin inhibited PAR2 and the malignant progression of lung cancer cells. Melittin increased the chemotherapeutic sensitivity of gefitinib in lung cancer by inhibiting PAR2.ConclusionPAR2 may promote the proliferation, invasion, and colony formation of lung cancer cells by promoting EMT. Patients with a high expression of PAR2 have a poor prognosis. Inhibition of PAR2 increased the chemotherapeutic sensitivity of gefitinib. PAR2 may be a potential therapeutic target and diagnostic marker for lung cancer.

Penetrance of Arsenic Modulates Gene Expression of Epithelial Mesenchymal Transition Marker SOX4, EpCAM and CK19 Genes Through MTHFR C677T Polymorphism Regulation in Circulating Tumour Cells Isolated from Breast Cancer Patients

Arsenic is a common environmental agent that modulates various kind of disease including cancer. However, the exact role of arsenic induce in breast cancer development remains unknown in circulating tumor cells (CTCs). Therefore, present study has been designed with the aims to elucidate the impact of arsenic exposure i.e 24, 48, and 72 hours on epithelial-mesenchymal transition (EMT) markers such as Sox4, EpCAM, and CK19. RT-PCR analysis was carried out for the characterization of EMT with specific primers on agarose gel electrophoresis. DNA copy number variation (DNACNVs) was evaluated to assess genetic susceptibility and MTHFR C677T gene polymorphism was examined to determine the “risk factor” in CTCs isolated from breast cancer patients. Interestingly, findings reveal the differential gene expression and a pattern of down regulation (under expression) were observed during 24 and 48 hours of exposure, followed by up regulation at 72 hours. Genetic heterogeneity of the MTHFR C→T allele was detected only at 48 hours, suggesting that arsenic increase “risk” for developing breast cancer by modulating EMT marker either alone or synergistically with MTHFR in CTCs.

Assessing Clinical Relevance of Gastric Cancer Cell Lines as Tumor Models by Identifying Key Genes and miRNAs involved in EMT Metastasis Pathway

Cancer cell lines are in-vitro cancer cell models that continue to divide and proliferate under controlled laboratory conditions. They are used in medical research to study tumor pathology and for testing therapeutics efficacy. Despite their extensive application, significant differences in genetic and transcriptomic expression exist between cell lines and tumor cells. The purpose of this study was to identify the relevance of gastric cancer cell lines as suitable models for gastric tumor research. Using a two-tiered bioinformatics approach- a comparison between the gastric tumor and the gastric cancer cell line was performed using functional genomics data available in the literature to identify the common Differentially Expressed Genes (DEGs). The study revealed differential expression of a total 380 genes common in both gastric cancer cell lines and tumor samples. Interestingly, four of these genes were identified as the Epithelial Mesenchymal Transition (EMT) markers, involved in the accelerated metastasis, of which two genes with discrete over-expression patterns, namely- ITGB1 and TGFB1 were selected for further analysis. Their survival analysis revealed their key role in gastric cancer severity, evident by the regressed survival rate of gastric cancer patients upon their overexpression. A total of nine tumor-suppressor miRNAs relevant to ITGB1 and TGFB1 were also identified. In conclusion, our findings indicate that inspite of various genetic and transcriptomic deviations, the tumor and cell line exhibit comparable expression patterns. The data obtained would be useful to implore the druggability of these targets with a goal of improved translational rates.

Noggin contributes to brain metastatic colonization of lung cancer cells

BackgroundBrain metastasis is a common complication among patients with lung cancer, yet the underlying mechanisms remain unclear. In this study, we aimed to investigate the pathogenesis of brain metastasis in lung cancer.MethodsWe established highly colonizing metastatic lung cancer cells, A549-M2, through multiple implantations of A549 human lung cancer cells in the carotid artery of athymic nude mice.ResultsCompared to parental cells (M0), M2 cells demonstrated slower growth in culture plates and soft agar, as well as lower motility and higher adhesion, key characteristics of mesenchymal–epithelial transition (MET). Further analysis revealed that M2 cells exhibited decreased expression of epithelial–mesenchymal transition markers, including ZEB1 and Vimentin. M2 cells also demonstrated reduced invasiveness in co-culture systems. RNA sequencing and gene set enrichment analysis confirmed that M2 cells underwent MET. Intriguingly, depletion of Noggin, a BMP antagonist, was observed in M2 cells, and replenishment of Noggin restored suppressed migration and invasion of M2 cells. In addition, Noggin knockdown in control M0 cells promoted cell attachment and suppressed cell migration, suggesting that Noggin reduction during brain colonization causes inhibition of migration and invasion of metastatic lung cancer cells.ConclusionsOur results suggest that lung cancer cells undergo MET and lose their motility and invasiveness during brain metastatic colonization, which is dependent on Noggin.

NXC736 Attenuates Radiation-Induced Lung Fibrosis via Regulating NLRP3/IL-1β Signaling Pathway.

Radiation-induced lung fibrosis (RILF) is a common complication of radiotherapy in lung cancer. However, to date no effective treatment has been developed for this condition. NXC736 is a novel small-molecule compound that inhibits NLRP3, but its effect on RILF is unknown. NLRP3 activation is an important trigger for the development of RILF. Thus, we aimed to evaluate the therapeutic effect of NXC736 on lung fibrosis inhibition using a RILF animal model and to elucidate its molecular signaling pathway. The left lungs of mice were irradiated with a single dose of 75 Gy. We observed that NXC736 treatment inhibited collagen deposition and inflammatory cell infiltration in irradiated mouse lung tissues. The damaged lung volume, evaluated by magnetic resonance imaging, was lower in NXC736-treated mice than in irradiated mice. NXC736-treated mice exhibited significant changes in lung function parameters. NXC736 inhibited inflammasome activation by interfering with the NLRP3-ASC-cleaved caspase-1 interaction, thereby reducing the expression of IL-1β and blocking the fibrotic pathway. In addition, NXC736 treatment reduced the expression of epithelial-mesenchymal transition markers such as α-SMA, vimentin, and twist by blocking the Smad 2,3,4 signaling pathway. These data suggested that NXC736 is a potent therapeutic agent against RILF.

INHBA gene silencing inhibits proliferation, migration, and invasion of osteosarcoma cells by repressing TGF-β signaling pathway activation

BackgroundOsteosarcoma (OS) is a refractory malignancy. This study aimed to explore the roles and mechanisms of Inhibin subunit beta A (INHBA) in OS.MethodsINHBA expression levels in OS tissues and cells were assessed using RT-qPCR and western blotting. The impact of INHBA silencing on OS development was then explored by transfecting the OS cell lines U2OS and MG63 with INHBA-small interfering RNA (siRNA). The influence of INHBA silencing on U2OS and MG63 cell proliferation, migration, and invasion was examined using MTT and Transwell assays. Epithelial–mesenchymal transition (EMT) markers (E-cadherin and N-cadherin) were analyzed by RT-qPCR. The expression of genes involved in cell proliferation, migration, invasion, and the TGF-β signaling pathway was evaluated by western blotting and RT-qPCR.ResultsINHBA levels were elevated in the OS tissues and cells. Furthermore, the transforming growth factor-β (TGF-β) signaling pathway of OS cells was suppressed in response to INHBA-siRNA, whereas proliferation, migration, and invasion of OS cells were inhibited. Besides, INHBA-siRNA significantly inhibited OS cell EMT, evidenced by enhanced E-cadherin mRNA expression and reduced N-cadherin mRNA expression. Further mechanistic studies revealed that the TGF-β1 agonist SRI-011381 hydrochloride increased OS cell proliferation, migration, and invasion after INHBA downregulation.ConclusionWe found that INHBA silencing could play a vital role in OS via TGF-β1-regulated proliferation, migration, and invasion.

Hydrogen peroxide enhances transforming growth factor beta-2 induced epithelial–mesenchymal transition of ARPE-19 cells

BackgroundIn the pathophysiology of the retina, the epithelial–mesenchymal transition and oxidative stress play key roles. To find a relationship between oxidative stress and diabetic retinopathy or proliferative retinopathy, we used an in-silico approach. We also used an in vitro investigation to examine the synergistic effect of transforming growth factors-β2 (TGFβ2) and hydrogen peroxide (H2O2) on the epithelial–mesenchymal transition (EMT) of ARPE-19 cells, analyzing cell survival, cell migration by wound healing, and expression epithelial–mesenchymal transition markers at the protein and mRNA levels.ResultsFor 24 h, human retinal pigment epithelial cells (ARPE-19) were cultured in standard conditions and subjected to various concentrations of TGFβ2, H2O2, and their combinations. According to a bioinformatics study, signaling pathways including hypoxia-inducible factor (HIF-1), forkhead box O (FoxO), phosphoinositide-3-kinase (PI3K-Akt), and transforming growth factors-β (TGFβ-signaling), both growth factors and oxidative stress-associated proteins play a significant role in the development of retinopathies. In vitro studies showed that oxidative stress induced by H2O2 and TGFβ2 enhances the epithelial–mesenchymal transition of ARPE-19 cells.ConclusionsHydrogen peroxide enhances the TGFβ2-mediated epithelial–mesenchymal transition of human retinal pigment epithelial cells, or at least in cell line ARPE-19 cells.Graphical abstract

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial–mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies.

Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy

Cancer-associated adipocytes (CAAs), one of the primary stromal components, exhibit intimate crosstalk and release multiple cell factors mediating local and systemic biological effects. However, the role of CAAs in the regulation of systemic immune responses and their potential value in the clinical treatment of triple-negative breast cancer (TNBC) are not well described. Transcriptome sequencing was performed on CAA and normal adipocyte (NA) tissues isolated from surgically resected samples from TNBC patients and healthy controls. Cytokines, including C-X-C motif chemokine ligand 8 (CXCL8, also known as IL-8), secreted from NAs and CAAs were compared by transcriptome sequencing and enzyme-linked immunosorbent assay (ELISA). Proliferation, migration and invasion assays were employed to analyze the role of CAAs and CAA-derived CXCL8 (macrophage inflammatory protein-2 (MIP2) as a functional surrogate in mice). TNBC syngraft models were established to evaluate the curative effect of targeting CXCL8 in combination with anti-PD-1 therapies. Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB), polymerase chain reaction (PCR) array, flow cytometry, immunohistochemistry (IHC), and immunofluorescence (IF) were applied to analyze immune cell infiltration and epithelial–mesenchymal transition (EMT) markers. Specifically, we demonstrated that CAAs and CAA-derived CXCL8 played important roles in tumor growth, EMT, metastasis and tumor immunity suppression. CAA-derived CXCL8 remodeled the tumor immune microenvironment not only by suppressing CD4+ T and CD8+ T immune cell infiltration but also by upregulating CD274 expression in TNBC. The combination of targeting the CXCL8 pathway and blocking the PD-1 pathway synergistically increased the tumor immune response and inhibited tumor progression. Thus, our results highlight the molecular mechanisms and translational significance of CAAs in tumor progression and immune ecosystem regulatory effects and provide a better understanding of the potential clinical benefit of targeting CAA-derived CXCL8 in antitumor immunity and as a new therapeutic moiety in TNBC.

2-Methoxyestradiol ameliorates paraquat-induced pulmonary fibrosis by inhibiting the TGF-β1/Smad2/3 signaling pathway

Paraquat (PQ) is a highly effective and highly toxic herbicide that is highly toxic to both humans and animals. Pulmonary fibrosis is the primary cause of fatality in patients with PQ poisoning, there is no effective drug treatment yet. 2-Methoxyestradiol (2ME) is a natural metabolite of estradiol with anti-tumor, anti-angiogenesis, and anti-proliferative effects. Whether 2ME has the potential to inhibit pulmonary fibrosis induced by PQ is unclear. This study aims to investigate the potential effects and mechanism of 2ME on PQ-induced pulmonary fibrosis. C57BL/6 mice and A549 cells were exposed to PQ to establish pulmonary fibrosis model. In vivo, Hematoxylin and eosin (H&E) staining was utilized to assess the pathological characteristics. Masson's trichrome staining was employed to evaluate the collagen deposition. Western blot and immunohistochemistry were conducted to determine the expressions of fibrosis markers. In vitro, the expressions of epithelial–mesenchymal transition (EMT) markers were detected using western blot and immunofluorescence to evaluated the potential inhibition of PQ-induced EMT by 2ME. And proteins associated with the TGF-β1/Smad2/3 signaling pathway were measured by western blot in vivo and in vitro. The result found that 2ME can ameliorated PQ-induced pulmonary fibrosis and inhibit the activation of TGF-β1/Smad2/3 signaling pathway. These findings suggest that 2ME may serve as a potential therapeutic agent for treating PQ-induced pulmonary fibrosis.

A Pharmacological Investigation of Eph-Ephrin Antagonism in Prostate Cancer: UniPR1331 Efficacy Evidence.

The Eph kinases are the largest receptor tyrosine kinases (RTKs) family in humans. PC3 human prostate adenocarcinoma cells are a well-established model for studying Eph-ephrin pharmacology as they naturally express a high level of EphA2, a promising target for new cancer therapies. A pharmacological approach with agonists did not show significant efficacy on tumor growth in prostate orthotopic murine models, but reduced distal metastasis formation. In order to improve the comprehension of the pharmacological targeting of Eph receptors in prostate cancer, in the present work, we investigated the efficacy of Eph antagonism both in vitro and in vivo, using UniPR1331, a small orally bioavailable Eph-ephrin interaction inhibitor. UniPR1331 was able to inhibit PC3 cells' growth in vitro in a dose-dependent manner, affecting the cell cycle and inducing apoptosis. Moreover, UniPR1331 promoted the PC3 epithelial phenotype, downregulating epithelial mesenchymal transition (EMT) markers. As a consequence, UniPR1331 reduced in vitro PC3 migration, invasion, and vasculomimicry capabilities. The antitumor activity of UniPR1331 was confirmed in vivo when administered alone or in combination with cytotoxic drugs in PC3-xenograft mice. Our results demonstrated that Eph antagonism is a promising strategy for inhibiting prostate cancer growth, especially in combination with cytotoxic drugs.

Integrative Analysis Reveals STC2 as a Prognostic Biomarker of Laryngeal Squamous Cell Carcinoma.

Stanniocalcin 2 (STC2) is involved in many tumour types, but it remains unclear what its biological function is in laryngeal squamous cell carcinoma (LSCC). Therefore, we investigated STC2's expression, potential function, and prognostic significance of in LSCC. The expression and prognosis of STC2 in LSCC were described using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. In the TCGA database, the relationship between STC2 and immune infiltration, expression of immune cell chemokine and receptor genes, immune cell molecular marker genes, and epithelial‒mesenchymal transition (EMT) marker genes were analysed. The biological processes involved in STC2 and its expression-related genes were analysed comprehensively using bioinformatics. The single-gene ceRNA network of STC2 was constructed in the TCGA database. Finally, LSCC patients' tumour tissue STC2 expression was verified. STC2 silencing with the RNAi technique was used for the determination of cellular functions in a laryngeal cancer cell line. STC2 expression was higher in most tumours, including LSCC, than in normal tissues and was associated with poor prognosis. The relative proportions of naïve B, plasma, follicular helper T, and macrophage M0 cells in LSCC and normal samples differed significantly. STC2 expression correlated significantly positively with that of TGFB1 (biomarker of Tregs) and significantly negatively with that of D79A and CD19 (biomarkers of B cells). Furthermore, STC2 affected chemokine and receptor gene expression in immune cells. STC2 expression correlated with EMT marker gene expression in LSCC. STC2 was enriched in the PI3K/AKT signalling pathway, extracellular matrix (ECM) organisation, ECM-receptor interaction, and other tumour-related signalling pathways. STC2 was highly expressed in our clinical samples. N-cadherin and vimentin expression were decreased in the TU686 cell line after successful silencing of STC2, indicating that high STC2 expression may prompt LSCC cells to adopt a mesenchymal cell phenotype. STC2 silencing substantially reduced proliferation and migration in the TU686 cell line. STC2 may be a promising predictive biomarker for tumours, providing new approaches for LSCC diagnosis and treatment monitoring.

Functional consequences of A-to-I editing of miR-379 in prostate cancer cells

Prostate cancer is the predominant cause of cancer in men, but there is still a lack of biomarkers and treatments for metastatic spread. The initial promise of microRNAs to provide avenues to solve these problems has been dampened by the realisation that microRNAs co-exist in multiple functionally distinct isoforms, for example due to A-to-I editing. We recently found that A-to-I-editing of microRNA-379 (miR-379) was associated with prostate cancer, and that only the unedited isoform was negatively correlated with aggressive disease. Here, we set out to decipher the biological effects of unedited and edited miR-379 in prostate cancer cells. After transfection of four different prostate cancer cell lines with isoform-specific miR-379 mimics, we performed assays for cell growth, colony formation, migration, cell–cell adhesion, and analysed epithelial–mesenchymal transition (EMT) and stemness markers. We found that unedited miR-379 affected cell growth, with a promoting function in androgen receptor (AR)-negative cells and an inhibiting effect in AR-positive cells. This is supported by our in silico analysis that found unedited miR-379 targets are predicted to be predominantly involved in cellular proliferation whereas the targets of edited miR-379 are not. We further found that both miR-379 isoforms could promote colony formation, migration, and cell–cell adhesion. Overall, our data suggests that editing of miR-379 attenuates the growth-suppressive function of unedited miR-379 in androgen-sensitive prostate cancer cells, thereby promoting tumor growth.