Epithelial-mesenchymal Transition Factors Research Articles

Enzymatic activity of HIV-1 protease defines migration of tumor cells in vitro and enhances their metastatic activity in vivo

Overexpression of aspartic proteases, as cathepsin D, is an independent marker of poor prognosis in breast cancer, correlated with the incidence of clinical metastasis. We aimed to find if HIV-1 aspartic protease (PR) can play a similar role. Murine adenocarcinoma 4T1luc2 cells were transduced with lentivirus encoding inactivated drug-resistant PR, generating subclones PR20.1 and PR20.2. Subclones were assessed for production of reactive oxygen species (ROS), expression of epithelial-mesenchymal transition (EMT) factors, and in vitro migratory activity in the presence or absence of antioxidant N-acetyl cysteine and protease inhibitors. Tumorigenic activity was evaluated by implanting cells into BALB/c mice and following tumor growth by calipering and bioluminescence imaging in vivo, and metastases, by organ imaging ex vivo. Both subclones expressed PR mRNA, and PR20.2, also the protein detected by Western blotting. PR did not induce production of ROS, and had no direct effect on cell migration rate, however, treatment with inhibitors of drug-resistant PR suppressed the migratory activity of both subclones. Furthermore, expression of N-cadherin and Vimentin in PR20.2 cells and their migration were enhanced by antioxidant treatment. Sensitivity of in vitro migration to protease inhibitors and to antioxidant, known to restore PR activity, related the effects to the enzymatic activity of PR. In vivo, PR20.2 cells demonstrated higher tumorigenic and metastatic activity than PR20.1 or parental cells. Thus, HIV-1 protease expressed in breast cancer cells determines their migration in vitro and metastatic activity in vivo. This effect may aggravate clinical course of cancers in people living with HIV-1.

SHP-1 mediates cigarette smoke extract-induced epithelial-mesenchymal transformation and inflammation in 16HBE cells.

Src-homology region 2 domain-containing phosphatase 1 (SHP-1) is considered an anti-inflammatory factor, but its role in chronic obstructive pulmonary disease (COPD) remains unknown. Herein, overexpression of SHP-1 was utilized to explore the functions of SHP-1 in COPD models established by stimulating 16HBE cells with cigarette smoke extracts (CSE) in vitro. SHP-1 was downregulated in both COPD patients and CES-treated 16HBE cells. SHP-1 overexpression reinforced cell viability and significantly prevented CSE-induced cell apoptosis in 16HBE cells. Furthermore, SHP-1 overexpression greatly reversed the CSE-induced migration, epithelial-mesenchymal transition (EMT), and pro-inflammatory factor production in 16HBE cells. In addition, CSE activated the P65 and PI3K/AKT pathways in 16HBE cells, which was also reversed by SHP-1 overexpression. Our findings indicated that SHP-1 alleviated CSE-induced EMT and inflammation in 16HBE cells, suggesting that SHP-1 regulated the development of COPD, and these functions may be linked to the inhibition of the PI3K/AKT pathway.

Significance of Runt-related transcription factor 1 and Notch1 expression in non-small-cell lung cancer: involvement in epithelial-mesenchymal transition and epidermal growth factor receptor-tyrosine kinase inhibitor therapy resistance

Objective One of the main obstacles to treating patients with non-small-cell lung cancers (NSCLC) is the emergence of drug resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Aim To investigate the prognostic relevance of Runt-related transcription factor 1 (RUNX1) and Notch1 in NSCLC and to evaluate their potential involvement in induction of epithelial-mesenchymal transition and resistance to EGFR-TKI therapy. Materials and methods Immunohistochemical study of RUNX1, Notch1, E-cadherin, and hypoxia-inducible factor 1α (HIF-1α) was conducted upon 83 cases diagnosed as NSCLC. The research was conducted in the departments of pathology, chest, and medical oncology of the Faculty of Medicine, Benha University. Results A significant relation was found between RUNX1 and sex (P=0.001), smoking history (P=0.002), and tumor grade (P=0.002). High RUNX1 expression was associated with poor OS and DFS (P=0.003 and 0.005), respectively. Cases with positive Notch1 expression were significantly associated with tumor grade (P=0.005) and tumor stage (P=0.024). A significant association was detected between Notch1 expression and poor OS and DFS (P=0.025 and 0.011), respectively. A statistically significant correlation was found between RUNX1 and Notch1 expressions (P=0.040). Moreover, high RUNX1 and positive Notch1 expressions were significantly associated with negative E-cadherin and positive HIF-1α expressions. Resistance against EGFR–TKI therapy was significantly associated with high RUNX1, positive Notch1, negative E-cadherin, and positive HIF-1α expressions, in EGFR-mutated cases. Conclusions RUNX1 and Notch1 may be involved in therapy resistance through the induction of epithelial–mesenchymal transition and may serve as prognostic markers in patients with NSCLC.

Energy stress-activated AMPK phosphorylates Snail1 and suppresses its stability and oncogenic function

Triple-negative breast cancer (TNBC) is a highly lethal malignancy with limited therapy options. Aberrant metabolism, a key hallmark of human cancers, plays a crucial role in tumor progression, therapeutic responses and TNBC-related death. However, the underlying mechanisms are not fully understood. In this study, we delineate a previously unrecognized role of aberrant glucose metabolism in regulating the turnover of Snail1, which is a key transcriptional factor of epithelial-mesenchymal transition (EMT) and critically contributes to the acquisition of stemness, metastasis and chemo-resistance. Mechanistically, we demonstrate that AMP-activated protein kinase (AMPK), when activated in response to glucose deprivation, directly phosphorylates Snail1 at Ser11. Such a phosphorylation modification of Snail1 facilitates its recruitment of the E3 ligase FBXO11 and promotes its degradation, thereby suppressing stemness, metastasis and increasing cellular sensitivity to chemotherapies in vitro and in vivo. Clinically, histological analyses reveal a negative correlation between p-AMPKα and Snail1 in TNBC specimens. Taken together, our findings establish a novel mechanism and functional significance of AMPK in linking glucose status to Snail1-dependent malignancies and underscore the potential of AMPK agonists as a promising therapeutic strategy in the management of TNBC.

Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/β-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

PSMA2 promotes glioma proliferation and migration via EMT

BackgroundGliomas advance rapidly and are associated with a poor prognosis. Epithelial-mesenchymal transition (EMT) accelerates the progression of gliomas, exerting a pivotal role in glioma development. Proteasome subunit alpha type-2 (PSMA2) exhibits high expression levels in gliomas. however, its specific involvement in glioma progression and its correlation with EMT remain elusive. This study aims to elucidate the role of PSMA2 in glioma progression and its potential association with EMT. MethodsOnline tools were employed to analyze the expression patterns and survival curves of PSMA2 in gliomas. The relationship between PSMA2 and various characteristics of glioma patients was investigated using data from the TCGA and CGGA databases. In vitro, cell proliferation and migration were assessed through CCK-8, colony formation, and transwell assays. Furthermore, a tumor xenograft model in nude mice was established to evaluate in vivo tumorigenesis. Protein binding to PSMA2 was scrutinized using co-immunoprecipitation MS (co-IP MS). The potential biological functions and molecular pathways associated with PSMA2 were explored through GO analysis and KEGG analysis, and the correlation between PSMA2 and EMT was validated through correlation analysis and Western blot experiments. ResultsBioinformatics analysis revealed a significant upregulation of PSMA2 across various cancers, with particularly heightened expression in gliomas. Moreover, elevated PSMA2 levels were correlated with advanced tumor stages and diminished survival rates among glioma patients. Inhibition of PSMA2 demonstrated a pronounced suppressive effect on glioma cell proliferation, both in vitro and in vivo. Knockdown of PSMA2 also impeded the migratory capacity of glioma cells. GO and KEGG enrichment analyses indicated that PSMA2-binding proteins (identified through Co-IP-MS) were associated with cell adhesion molecule binding and cadherin binding. Western blot results further confirmed the role of PSMA2 in promoting epithelial-mesenchymal transition (EMT) in glioma cells. ConclusionOur study provides evidence supporting the role of PSMA2 as a regulatory factor in EMT and suggests its potential as a prognostic biomarker for glioma progression.

NcRNAs Orchestrate Chemosensitivity Induction by Neddylation Blockades.

We performed an integrative transcriptomic in silico analysis using lung adenocarcinoma A549 cells treated with the neddylation inhibitor MLN4924 and the gefitinib-resistant PC9 cell line (PC9GR). We focused on the transcriptional effects of the top differentially expressed ncRNA biotypes and their correlating stemness factors. Interestingly, MLN4924-treated cells showed a significant upregulation of mRNAs involved in carcinogenesis, cell attachment, and differentiation pathways, as well as a parallel downregulation of stemness maintenance and survival signaling pathways, an effect that was inversely observed in PC9GR cells. Moreover, we found that stemness factor expression could be contrasted by selected up-regulated ncRNAs upon MLN4924 treatment in a dose and time-independent manner. Furthermore, upregulated miRNAs and lncRNA-targeted mRNAs showed an evident enrichment of proliferation, differentiation, and apoptosis pathways, while downregulated ncRNA-targeted mRNAs were implicated in stem cell maintenance. Finally, our results proved that stemness (KLF4 and FGFR2) and epithelial-mesenchymal transition (ZEB2, TWIST2, SNAI2, CDH2, and VIM) factors, which are highly expressed in PC9GR cells compared to gefitinib-sensitive PC9 cells, could be abrogated with the neddylation inhibitor MLN4924 mainly through activation of epithelial differentiation pathways, thus exerting a protective role in lung cancer cells and chemosensitivity against lung tumorigenic transformation.

The effect of microRNA-9 overexpression on inhibition of melanoma cancer stem cells tumorigenicity.

Most of the studies have reported the downregulation of miR-9 in metastatic melanomas compared to primary tumors. They indicated that miR-9 negatively regulates the epithelial-to-mesenchymal transition (EMT) by inhibiting SNAIL1 expression and consequently promotes CDH1 expression. Since the process of EMT is associated to stem cell features, it could be interesting to study the effect of miR-9 on melanoma cancer stem cells. In the present study, we examined the effects of miR-9 manipulation on the stemness potential of melanoma cells. Our data demonstrated that the overexpression of miR-9 in A375 and NA8 cells significantly inhibits the ability of proliferation, self-renewal, migration, and tumorigenicity of melanoma cells which was concomitant with changes in the level of BRAF , some EMT factors, and stemness genes. Likewise, the reduction of miR-9 levels led to an increase in cell proliferation, colony and sphere formation, and the ability of cell migration and tumorigenicity. In conclusion, our results specified the role of miR-9 as a tumor suppressor miRNA to inhibit many aspects of melanoma stem cells, and therefore, it could be a potential candidate for the suppression of melanoma growth and progression.

Insight into the differential toxicity of PFOA and PFBA based on a 3D-cultured MDA-MB-231 cell model

Perfluoroalkyl substances (PFASs) are a category of high-concerned emerging contaminants which are suspected to correlate with various human adverse health outcomes including tumors. It is also a question whether short-chain PFASs are qualified alternatives under the regulation of long-chain PFASs. In this study, a three-dimensional (3D) culture system based on Gelatin methacrylate (GelMA) hydrogel matrix was used to investigate the impacts of 120-h perfluorooctanoic acid (PFOA) and perfluorobutanoic acid (PFBA) exposure of MDA-MB-231 cells. The results showed that PFOA exposure promoted the proliferation, migration, and invasion of MDA-MB-231 cells in an environmentally relevant concentration range (0.1 to 10 μM), exhibiting a clear malignant-promoting risk. In contrast, PFBA only showed a trend to induce non-invasive cell migration. Hippo/YAP signaling pathway was identified as the contributor to the differences between the two PFASs. PFOA but PFBA reduced YAP phosphorylation and increased the nuclear content of YAP, which further facilitated abundant key factors of epithelial-mesenchymal transition (EMT) process. Our results provided a new idea for the carcinogenicity of PFOA using a 3D-based paradigm. Although the effects by PFBA were much milder than PFOA in the current test duration, the cell model suitable for longer exposure is still necessary to better assess the safety of alternative short-chain PFASs.

Shake It Up Baby Now: The Changing Focus on TWIST1 and Epithelial to Mesenchymal Transition in Cancer and Other Diseases.

TWIST1 is a transcription factor that is necessary for healthy neural crest migration, mesoderm development, and gastrulation. It functions as a key regulator of epithelial-to-mesenchymal transition (EMT), a process by which cells lose their polarity and gain the ability to migrate. EMT is often reactivated in cancers, where it is strongly associated with tumor cell invasion and metastasis. Early work on TWIST1 in adult tissues focused on its transcriptional targets and how EMT gave rise to metastatic cells. In recent years, the roles of TWIST1 and other EMT factors in cancer have expanded greatly as our understanding of tumor progression has advanced. TWIST1 and related factors are frequently tied to cancer cell stemness and changes in therapeutic responses and thus are now being viewed as attractive therapeutic targets. In this review, we highlight non-metastatic roles for TWIST1 and related EMT factors in cancer and other disorders, discuss recent findings in the areas of therapeutic resistance and stemness in cancer, and comment on the potential to target EMT for therapy. Further research into EMT will inform novel treatment combinations and strategies for advanced cancers and other diseases.

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Cytokines as drivers: Unraveling the mechanisms of epithelial-mesenchymal transition in COVID-19 lung fibrosis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like other viruses, can induce proliferation of myofibroblasts and even lead to fibrosis in the lung. Epithelial-mesenchymal transition (EMT) is thought to play an essential role in the pathogenesis of Coronavirus disease 19 (COVID-19). EMT is originally a critical process that regulates the development of different tissues in the embryo, but in inflammatory situations, EMT tries to be activated again to control inflammation or even heal inflammatory damage. However, in pathological situations, such as chronic viral infections (e.g., COVID-19) or pulmonary fibrosis initiation, this benign healing transforms into sinister nature, pushing the lung into the fibrotic process. Notably, the cytokines released by inflammatory cells and the chronic inflammatory microenvironment shared by fibrotic cells promote each other as critical factors in the induction of pathological EMT. In the induction of SARS-CoV-2 virus, cytokines are an essential mediator of EMT transformation, and a summary of whether COVID-19 patients, during the infection phase, have many persistent inflammatory mediators (cytokines) that are a causative factor of EMT has not yet appeared.The following common signaling drivers, including Transforming growth factor beta (TGF-β), cytokines, Notch signaling pathway, Wnt and hypoxia signaling pathways, drive the regulation of EMT. In this review, we will focus on 3 key EMT signaling pathways: TGF-β, Leucine zipper transcription factor like 1 (LZTFL1) and the common interleukin family expressed in the lung. TGF-β-induced SNAIL and LZTFL1 were identified as regulatory EMT in COVID-19. For cytokines, the interleukin family is a common inducer of EMT and plays an essential role in the formation of the microenvironment of fibrosis. We sought to demonstrate that cytokines act as "communicators" and build the "microenvironment" of fibrosis together with EMT as a "bridge" to induce EMT in fibrosis. The mechanisms utilized by these two pathways could serve as templates for other mesenchymal transformations and provide new potential therapeutic targets.

PFKFB3 promotes endometriosis cell proliferation via enhancing the protein stability of β-catenin

Endometriosis is a common inflammatory disease in women of reproductive age and is highly associated with infertility. However, the molecular mechanism of endometriosis remains unclear. 6-Phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a key enzyme in glycolysis and plays an important regulatory role in the development of cancer. Here we found that PFKFB3 is highly expressed in endometriotic tissues. PFKFB3 promotes the proliferation and growth of endometriosis cells. Meanwhile, PFKFB3 promotes glycolysis in endometriosis cells. Furthermore, PFKFB3 promotes migration and invasion of endometriosis cells. On this basis, we found that PFKFB3 promotes epithelial-mesenchymal transition (EMT) in endometriosis cells. PFKFB3 interacts with the essential factor of EMT, β-catenin, and promotes the protein stability of β-catenin. In addition, the PFKFB3 inhibitor PFK-015 inhibites the growth of endometriosis cells and the development of endometrial tissue. In conclusion, our study shows that PFKFB3 plays an important role in the development of endometriosis and provides new ideas for the clinical diagnosis or treatment of endometriosis.

A unique cell population expressing the Epithelial-Mesenchymal Transition-transcription factor Snail moderates microglial and astrocyte injury responses.

Insults to the central nervous system (CNS) elicit common glial responses including microglial activation evidenced by functional, morphological, and phenotypic changes, as well as astrocyte reactions including hypertrophy, altered process orientation, and changes in gene expression and function. However, the cellular and molecular mechanisms that initiate and modulate such glial response are less well-defined. Here we show that an adult cortical lesion generates a population of ultrastructurally unique microglial-like cells that express Epithelial-Mesenchymal Transcription factors including Snail. Knockdown of Snail with antisense oligonucleotides results in a postinjury increase in activated microglial cells, elevation in astrocyte reactivity with increased expression of C3 and phagocytosis, disruption of astrocyte junctions and neurovascular structure, increases in neuronal cell death, and reduction in cortical synapses. These changes were associated with alterations in pro-inflammatory cytokine expression. By contrast, overexpression of Snail through microglia-targeted an adeno-associated virus (AAV) improved many of the injury characteristics. Together, our results suggest that the coordination of glial responses to CNS injury is partly mediated by epithelial-mesenchymal transition-factors (EMT-Fsl).

Hepatoma Derived Growth Factor (HDGF) Targeting as a Therapeutic Approach for Radio-Naive and Radioresistant Hepatocellular Carcinoma

The role of radiotherapy in treating hepatocellular carcinoma (HCC) has gained importance. Hepatoma-derived growth factor (HDGF) is a nuclear growth factor involved in the development of HCC and its overexpression has been linked to tumor progression and radiation treatment failure. The aim of this study is to investigate the potential therapeutic benefits of HDGF-targeting treatment in combination with or without radiation for both radio-naive and radioresistant HCC cells. A series of radiation doses (2Gy, 4Gy, 8Gy, 12Gy, and 20Gy in one fraction) were administered to Huh7 human HCC cells. The gene expression profiles of radiation-treated Huh7 cells were analyzed. The radioresistant Huh7 cell line (Huh7-IR) was established by repeatedly irradiating with 2-week intervals with 8 Gy of X-ray to a total dose of 80 Gy. After acquiring radioresistance, the expression levels of HDGF, cancer stem cell (CSC) markers, and epithelial-mesenchymal transition (EMT) factors were validated. The pShuttle-shHDGF plasmid with adenovirus vector was generated for HDGF targeting treatment (HDGF knockdown). The effect of the HDGF targeting treatment on tumorigenic processes and cell cycle distribution in the radio-naive and radioresistant cells with or without irradiation were explored. After radiation treatment, qPCR analysis showed that the mRNA expression of HDGF, CSC markers (CD133, CD44, and ABCG2), and EMT markers (TWIST and SNAIL) increased over time, with the most significant upregulation observed between 48-72 hours. In the radioresistant Huh7-IR cells, HDGF, CSC markers (CD133 and CD44), and the EMT transcriptional factor (TWIST) were significantly higher than in the parental Huh7 cells. The Huh7-IR cells also showed a significant arrest in the G0/G1 phase. X-ray radiation alone significantly reduced cell proliferation and invasion in the Huh7 cells compared to the control group. The HDGF targeting treatment significantly enhanced the X-ray-mediated inhibition of colony formation and cell invasion in both the Huh7 and Huh7-IR cells. Targeting HDGF leads to cell cycle arrest (G2/M phase) in Huh7 and Huh7-IR cells. The HDGF targeting treatment in combination with X-ray irradiation results in extensive G2/M phase arrest. These results indicate the crucial role of HDGF in radiation therapy for radio-naive and radioresistant HCC cells. Targeting HDGF effectively suppressed colony formation and invasion, and enhanced cell cycle arrest in the G2/M phase in combination with radiation. HDGF may serve as a promising therapeutic target for improving HCC management strategies. Further investigation is required to fully comprehend the role of HDGF in improving radiation therapy efficacy for HCC.

Abstract PO-100: Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway

Abstract Introduction The enrichment of Fusobacterium nucleatum has been reported in both oral rinses and lesion samples from oral squamous cell carcinoma (OSCC) patients based on several case-control sequencing analyses, but studies on the potential role of F. nucleatum in OSCC tumorigenesis are still limited. In this study, we sought to elucidate the relationship between F. nucleatum and host transcriptome dysregulation in potentially carcinogenic pathways. Method The association between microbiota dysbiosis and host gene transcriptome was profiled by bacterial 16S rRNA V3-V4 amplicon sequencing and host gene long non-coding RNA sequencing (lncRNA-seq) for tumor and adjacent normal (AN) tissues from OSCC patients. Cell migration and invasion assays were performed using the in vitro bacteria-cell co-culture model. EMT-related molecular mechanisms induced by F. nucleatum were further validated by IncRNA-seq, real-time PCR, and western blot for bacteria-infected cells, respectively, at the genetic and proteomic levels. Result Significant enrichment of Fusobacterium was observed in OSCC tumor samples when compared to the paired AN samples. Correlation analysis between mucosal oral bacteria and host transcriptome in the OSCC tumor microenvironment elucidated the positive relationship between Fusobacterium and EMT pathways including significantly upregulated genes INHBA, SNAI2, and LAMC2. Using in-vitro cell models, we further observed that F. nucleatum significantly promoted cell migration and invasion, and significantly dysregulated the expression of several EMT factors including the increased inducer inhibin beta A (INHBA), core regulators SNAI1 (snai1) and SLUG (snai2), effectors vimentin (VIM) and N-cadherin (CDH2), and decreased E-cadherin (CDH1). Following the INHBA/SMAD pathway induced by F. nucleatum infection, the expression of an essential extracellular matrix glycoprotein, Laminin 332, was dramatically enhanced, which may trigger the phosphorylation of phosphatidylinositol 3-kinase and AKT kinase in the downstream PI3K/AKT pathway for the promoted cell migration and invasion ability. Conclusion Our study confirms the enrichment of Fusobacterium in the OSCC tumor microenvironment and reveals that F. nucleatum infection induces the INHBA/SAMD pathway and subsequently increases Laminin 332 expression, which may further promote the phosphorylation of PI3K/AKT and enhance OSCC migration and invasion. Citation Format: Hengyan Zhu, Liuyang Cai, Zigui Chen, Jason Y. K. Chan. Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-100.

Multilevel chitosan-gelatin particles loaded with P4HA1 siRNA suppress glioma development.

It has been reported that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) promoted tumor growth and metastasis of glioma; thus, targeting P4HA1 may be a promising therapeutic strategy against glioma. In consideration of the instability of siRNA in vivo, the chitosan-gelatin microspheres loaded with P4HA1 siRNA (P4HA1 siRNA@CGM) were employed. Firstly, the gel electrophoresis and hemolytic test were performed to assess the stability and blood compatibility of P4HA1 siRNA@CGM. Then, methyl thiazolyl tetrazolium (MTT), cell colony formation, Transwell assay, wound healing assay, gliosphere formation, tube formation, and Western blot were performed to assess the effects of P4HA1 siRNA@CGM on the biological functions of glioma. Finally, 125I-labeled P4HA1 siRNA@CGM was injected into the xenograft mice, radionuclide imaging was recorded, Ki67 and terminal deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining was performed to assess the effects of P4HA1 siRNA@CGM on tumor growth and apoptosis of glioma in vivo. The results showed that P4HA1 siRNA and P4HA1 siRNA@CGM not only markedly inhibited the proliferation, metastasis, gliosphere formation, and the protein levels of interstitial markers (N-cadherin and vimentin) and the transcription factors of epithelial-mesenchymal transition (EMT) (Snail, Slug, and Twist1) in glioma cells, but also inhibited the tube formation in human brain microvascular endothelial cells (HBMECs), and P4HA1 siRNA@CGM exhibited the better inhibitory effects than P4HA1 siRNA. Above results suggested the feasibility of P4HA1 siRNA@CGM in the clinical treatment of glioma.

Modulatory effects of point-mutated IL-32θ (A94V) on tumor progression in triple-negative breast cancer cells.

Breast cancer is a frequently diagnosed cancer and the leading cause of death among women worldwide. Tumor-associated macrophages stimulate cytokines and chemokines, which induce angiogenesis, metastasis, proliferation, and tumor-infiltrating immune cells. Although interleukin-32 (IL-32) has been implicated in the development and modulation of several cancers, its function in breast cancer remains elusive. Mutation of interleukin-32θ (IL-32θ) in the tissues of patients with breast cancer was detected by Sanger sequencing. RT-qPCR was used to detect the mRNA levels of inflammatory cytokines, chemokines, and mediators. The secreted proteins were detected using respective enzyme-linked immunosorbent assays. Evaluation of the inhibitory effect of mutant IL-32θ on proliferation, migration, epithelial-mesenchymal transition (EMT), and cell cycle arrest in breast cancer cells was conducted using MTS assays, migration assays, and Western blotting. A point mutation (281C>T, Ala94Val) was detected in IL-32θ in both breast tumors and adjacent normal tissues, which suppressed the expression of pro-inflammatory factors, EMT factors, and cell cycle related factors. Mutated IL-32θ inhibited the expression of inflammatory factors by regulating the NF-κB pathway. Furthermore, mutated IL-32θ suppressed EMT markers and cell cycle related factors through the FAK/PI3K/AKT pathway. It was inferred that mutated IL-32θ modulates breast cancer progression. Mutated IL-32θ (A94V) inhibited inflammation, EMT, and proliferation in breast cancer by regulating the NF-κB (p65/p50) and FAK-PI3K-GSK3 pathways.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

EMT Features in Claudin-Low versus Claudin-Non-Suppressed Breast Cancers and the Role of Epigenetic Modifications.

Breast cancers are heterogeneous and are classified according to the expression of ER, PR and HER2 receptors to distinct groups with prognostic and therapeutic implications. Within the triple-negative group, with no expression of these three receptors, molecular heterogeneity exists but is currently not exploited in the clinic. The claudin-low phenotype is present in a subset of triple-negative breast cancers and constitutes together with basal-like cancers the most extensive groups within triple-negative breast cancers. Suppression of epithelial cell adhesion molecules in claudin-low cancers is also a hallmark of Epithelial Mesenchymal Transition (EMT). The groups of claudin-low and claudin-non-suppressed breast cancers from the extensive publicly available genomic cohorts of the METABRIC study were examined to delineate and compare their molecular landscape. Genetic and epigenetic alterations of key factors involved in EMT and potentially associated with the pathogenesis of the claudin-low phenotype were analyzed in the two groups. Claudin-low cancers displayed up-regulation of several core transcription factors of EMT at the mRNA level, compared with claudin-non-suppressed breast cancers. Global promoter DNA methylation was increased in both groups of triple-negative cancers and in claudin-low ER-positive cancers compared with the rest of ER-positive cancers. Histone modifier enzymes, including methyltransferases, demethylases, acetyltransferases and deacetylases displayed amplifications more frequently in claudin-non-suppressed triple-negative cancers than in claudin-low counterparts and the expression of some of these enzymes differed significantly between the two groups. Claudin-low and claudin-non-suppressed triple-negative breast cancers differ in their landscape of EMT core regulators and epigenetic regulators. These differences may be explored as targets for therapeutic interventions specific to the two groups of triple-negative breast cancers.