Induction Of Epithelial-mesenchymal Transition Research Articles

In silico investigation of cannabinoids from Cannabis sativa leaves as a potential anticancer drug to inhibit MAPK-ERK signaling pathway and EMT induction

Genes related to MAPK-ERK signaling pathways, and epithelial-mesenchymal transition induction is evolutionarily conserved and has crucial roles in the regulation of important cellular processes, including cell proliferation. In this study, six cannabinoids from Cannabis sativa were docked with MAPK-ERK signaling pathways to identify their possible binding interactions. The results showed that all the cannabinoids have good binding affinities with the target proteins. The best binding affinities were MEK- tetrahydrocannabinol (– 8.8 kcal/mol) and P13k-cannabinol (– 8.5 kcal/mol). The root mean square deviation was calculated and used two alternative variants (rmsd/ub and rmsd/lb) and the values of rmsd/lb fluctuated 8.6–2.0 Å and for rmsd/ub from 1.0 to 2.0 Å that suggests the cannabinoids and protein complex are accurate and cannot destroy on binding. The study analyzed the pharmacokinetic and drug-likeness properties of six cannabinoids from C. sativa leaves using the SwissADME web tool. Lipinski's rule of five was used to predict drug-likeness and showed that all compounds have not violated it and the total polar surface area of cannabinoids was also according to Lipinski's rule that is benchmarked of anticancer drugs. Cannabinoids are meet the requirements of leadlikeness and synthetic accessibility values showed they can be synthesized. The molecular weight, XLOGP3, solubility (log S), and flexibility (FLEX) are according to the bioavailability radar. The bioavailability score and consensus Log Po/w fall within the acceptable range for the suitable drug. Pharmacokinetics parameters showed that cannabinoids cannot cross the blood–brain barrier, have high GI absorption as well as cannabinoids are substrates of (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) but no substrate of P-glycoprotein. Based on these findings, the study suggests that cannabinoids are suitable drugs that could be used as effective inhibitors for target proteins involved in cancer pathways. Among the six cannabinoids, cannabinol and tetrahydrocannabinol exerted maximum binding affinities with proteins of MAPK-ERK signaling pathways, and their pharmacokinetics and drug-likeness-related profiles suggest that these cannabinoids could be superlative inhibitors in cancer treatment. Further in vitro, in vivo, and clinical studies are needed to explore their potential in cancer treatment.

The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-β1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

Modulation of epithelial-mesenchymal transition by gemcitabine: Targeting ionizing radiation-induced cellular senescence in lung cancer cell

Ionizing radiation, a standard therapeutic approach for lung cancer, often leads to cellular senescence and the induction of epithelial-mesenchymal transition (EMT), posing significant challenges in treatment efficacy and cancer progression. Overcoming these obstacles is crucial for enhancing therapeutic outcomes in lung cancer management. This study investigates the effects of ionizing radiation and gemcitabine on lung cancer cells, with a focus on induced senescence, EMT, and apoptosis. Human-derived A549, PC-9, and mouse-derived Lewis lung carcinoma cells exposed to 10 Gy X-ray irradiation exhibited senescence, as indicated by morphological changes, β-galactosidase staining, and cell cycle arrest through the p53-p21 pathway. Ionizing radiation also promoted EMT via TGFβ/SMAD signaling, evidenced by increased TGFβ1 levels, altered EMT marker expressions, and enhanced cell migration. Gemcitabine, a first-line lung cancer treatment, was shown to enhance apoptosis in senescent cells caused by radiation. It inhibited cell proliferation, induced mitochondrial damage, and triggered caspase-mediated apoptosis, thus mitigating EMT in vitro. Furthermore, in vivo studies using a lung cancer mouse model revealed that gemcitabine, combined with radiation, significantly reduced tumor volume and weight, extended survival, and suppressed malignancy indices in irradiated tumors. Collectively, these findings demonstrate that gemcitabine enhances the therapeutic efficacy against radiation-resistant lung cancer cells, both by inducing apoptosis in senescent cells and inhibiting EMT, offering potential improvements in lung cancer treatment strategies.

The Evaluation of the N-cadherin Promoter’s ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy.
 To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-β and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and β-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test.
 After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of β-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-β in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion.
 Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/β-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/β-catenin in TNBC progression. Wnt/β-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT.Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/β-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/β-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

P53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

Wnt5a/β-catenin-mediated epithelial-mesenchymal transition: a key driver of subretinal fibrosis in neovascular age-related macular degeneration

BackgroundNeovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial–mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD.MethodsIn vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch's membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFβ1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The changes in EMT- and Wnt-related signaling molecules, as well as cell functions were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay or transwell migration assay. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8.ResultsThe in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of β-catenin in laser-induced CNV mice, as evidenced by the significant reduction in areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active β-catenin labeling. In vitro, Wnt5a/ROR1, active β-catenin, and some other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active β-catenin, as well as the EMT in TGFβ1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells.ConclusionsOur study reveals a reciprocal activation between Wnt5a and β-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.

Cuproptosis-Related Gene FDX1 Suppresses the Growth and Progression of Colorectal Cancer by Retarding EMT Progress.

Colorectal cancer (CRC) is a usual cancer and a kind of lethiferous cancer. Cuproptosis-related gene ferredoxin 1 (FDX1) has been discovered to act as a suppressor, thereby suppressing some cancers' progression. But, the regulatory functions of FDX1 in CRC progression keep vague. In this work, at first, through TCGA database, it was revealed that FDX1 exhibited lower expression in COAD (colon adenocarcinoma) tissues, and CRC patients with lower FDX1 expression had worse prognosis. Furthermore, FDX1 expression was verified to be down-regulated in CRC tissues (n = 30) and cells. It was further uncovered that FDX1 expression was positively correlated with CDH1 and TJP1 (epithelial marker), and negatively correlated with CDH2, TWIST1, and FN1 (stromal marker), suggesting that FDX1 was closely associated with the epithelial-mesenchymal transition (EMT) progress. Next, it was demonstrated that overexpression of FDX1 suppressed cell viability, invasion, and migration in CRC. Furthermore, it was verified that FDX1 retarded the EMT progress in CRC. Lastly, through rescue assays, the inhibited CRC progression mediated by FDX1 overexpression was rescued by EGF (EMT inducer) treatment. At last, it was uncovered that the tumor growth and metastasis were relieved after FDX1 overexpression, but these changes were reversed after EGF treatment. In conclusion, FDX1 inhibited the growth and progression of CRC by inhibiting EMT progress. This discovery hinted that FDX1 may act as an effective candidate for CRC treatment.

Abstract 211: Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer

Abstract Metastatic Breast Cancer is accountable for over half a million deaths worldwide each year, the majority of which are from the triple negative breast cancer (TNBC) subtype, known to be highly aggressive, metastatic and resistant to targeted therapy. Metastasis is driven by the ability of cancer cells to undergo epithelial to mesenchymal transition (EMT), a cell state transition involving a gain of stemness, cell motility and chemoresistance. EMT has been well characterized using a range of techniques based upon quantitative proteomics and transcription profiling, yet targeting EMT to prevent tumor metastasis and chemoresistance is an obstacle that remains to be fully overcome. Using a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS), we have measured changes in protein stability upon induction of EMT and have uncovered key enzymes in the citric acid (TCA) cycle that are stabilized during EMT of human mammary epithelial cells. Such enzymes are responsible for the conversion of TCA cycle intermediates required for a recently discovered post-translational modification. In TNBC cells, we find that loss of these enzymes causes not only global changes to crucial post-translational modification of mitochondrial proteins, but very specific alterations in several cytoskeletal proteins, leading to altered EMT status as well as reduced tumor growth and initiation both in vitro and in vivo. This demonstrates the importance of TCA cycle-controlled post-translational modification of cytoskeletal proteins in tumorigenesis and establishes basis for further investigation into their role in EMT and metastasis of breast cancer. Through pharmacological inhibition and immunofluorescence imaging, we discovered regulators of post-translational modifications that may be viable drug targets to overcome chemoresistance and relapse in TNBC patients with limited and inefficacious options. Citation Format: Jessica A. Lidster, Ser Yue Loo, Zhengwei Wu, Lingyun Dai, Seow Qi Ng, Nayana Prabhu, Dennis Kappei, Wai Leong Tam. Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer [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 211.

Abstract 5153: Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics

Abstract Background: Colorectal cancer (CRC) exhibits a heightened presence of intratumoral bacteria, and deficient DNA mismatch repair (dMMR) contributes to nucleotide mismatch accumulation, fostering mutagenesis. However, the combined impact of intratumoral bacteria and dMMR on the tumor immune microenvironment (TIME) in CRC, and its consequential influence on patient prognosis, remains elusive. Methods: We utilized samples from the Cancer Microbiome Atlas project (TCMA), collecting tumor and matched paracancer tissues from colorectal cancer patients. Phyloseq in R was employed to scrutinize microbial abundance differences. The effects of microbial enrichment on prognosis were assessed through survival analysis, and GSEA enrichment analysis provided mechanistic insights. Results: A total of 173 samples, including 28 dMMR tumors, 112 proficient MMR (pMMR) tumors, and 22 paired para-cancer tissues, were collected. Following stringent filtration, we analyzed 230 bacterial genera. Notably, 26 genera, including Fusobacterium and Streptococcus, showed significant enrichment in dMMR tumors compared to para-cancer tissues. Furthermore, 18 genera were enriched in dMMR tumors compared to pMMR tumors. Of these, 8 genera like Desulfovibrio and Pyramidobacter consistently exhibited enrichment in dMMR CRC compared to either para-cancer tissues or pMMR tumors. Survival analysis highlighted Pyramidobacter as the only genus significantly associated with prognosis in dMMR CRC. Increased Pyramidobacter abundance correlated with decreased overall survival (OS) (P value: 0.049). Stratifying dMMR CRC patients based on Pyramidobacter abundance further emphasized the significantly worse survival in the Pyramidobacter-High group (HR 0.93, 95%CI 1.03-9.60, log-rank p = 0.015). Exploring the impact of Pyramidobacter infection on tumor progression, we assessed TIME and oncogenic pathways between Pyramidobacter-Low and Pyramidobacter-High groups. While overall immune infiltration was higher in the Pyramidobacter-High group, it primarily stemmed from increased B cell and naive CD8+ T cell infiltration. Importantly, pathways enriched in the Pyramidobacter-High group included inflammatory response, epithelial-mesenchymal transition (EMT), and KRAS signaling, while those enriched in the Pyramidobacter-Low group encompassed IFNα response, IFNγ response, and DNA damage repair. These findings suggest Pyramidobacter's role in inducing inflammation, promoting tumor proliferation and metastasis, and suppressing interferon response. Conclusion: Pyramidobacter emerges as a key player in the development of dMMR CRC, exerting a pivotal influence on patient prognosis through the induction of inflammatory responses and EMT. Citation Format: Shisen LI, Yunlong LI, Hongjiang Ma, Shihao Qin, Feilong Zhao, Jipeng Li. Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics [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 5153.

Abstract 1444: Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer characterized by very poor survival outcomes. Genomic sequencing of human PDAC tissues and cell lines have identified four molecular subtypes: quasimesenchymal (QM), pancreatic progenitor, immunogenic, and aberrantly differentiated endocrine exocrine (ADEX). QM-PDAC has the worse prognosis, but no in vivo models exist for this molecular subtype. Our preliminary analysis using publicly available data demonstrated that the epithelial-to-mesenchymal transition (EMT) transcription factor TWIST1 is upregulated in QM-PDAC. We hypothesized that the TWIST1-dependent plasticity program was a key regulator of QM-PDAC subtype development. We created a tetracycline-inducible tissue specific Twist1 PDAC genetically engineered mouse model (GEMM). Tumor development in the mouse pancreatic ductal epithelium was directed using the Pdx1 promoter-Cre (P) allele leading to stable native expression of the LSL-KrasG12D oncogene (R) and LSL-rtTA-IRES-GFP (G) alleles. The non-Twist1 GEMM (PGR) was crossed with the Twist1-tetO-Luc (T) mouse to create the PGRT model which allows inducible expression of Twist1 and luciferase in pancreatic epithelium under doxycycline treatment (Tet-ON system). Tumor progression, lineage tracing and metastatic invasion are followed by fluorescence (GFP) and bioluminescence (BLI) imaging. The characterization of this novel PGRT mouse model showed decreased overall survival compared to control non-Twist1 containing mice (~35 weeks PGRT (n=17) vs ~60 weeks PGR (n=10)). Histological and immunohistochemistry analyses showed the PGRT pancreatic tumors resemble the QM-PDAC subtype and vimentin overexpression correlates with Twist1-dependent induction of EMT. We attempted to confirm the QM-PDAC profile via cellular markers: GATA6-, HNF1A-, KRT81+, KRT17+, KRT20+, P63+ and ∆P63+. The PGR and standard KPC (Kras+/LSL-G12D; Trp53+LSL-R172H; Pdx1-Cre) PDAC model were also profiled. We observed that 75% of the PGRT tumors displayed a QM-PDAC profile, contrasting with the absence of such profile in both PGR and KPC tumors. Examining the tumors from PGR and KPC, 100% of the PGR and 72.27% KPC tumors exhibited a non-squamous PDAC profile while an intermediate squamous profile was observed in 25% PGRT tumors and 27.27% KPC tumors. Furthermore, Twist1 induction resulted in intra-abdominal and lung metastases. We observed that 87.5% of the PGRT mice were positive for metastases while we observed no positivity for PGR mice and 63.64% positivity in KPC mice. In conclusion, these data suggest that Twist1 overexpression cooperates with KrasG12D for PDAC tumor development mimicking the human QM-PDAC subtype to promote progression towards metastasis and early lethality. Potential uses of this novel model include testing treatment strategies to ultimately improve patient outcomes with QM-PDAC. Citation Format: Muhammad Ajmal Khan, Jinhee Chang, Triet Nguyen, Danielle N. Council, Dipanwita Dutta Chowdhury, Amol Shetty, Yang Song, Aaron Chan, Christine Lam, Priyanshi Patel, Lucas Tran, Jiayu Chen, Katriana Nugent, Matthew Ballew, Ghali Lemtiri-Chlieh, Francesca A. Carrieri, Reem Malek, Hailun Wang, Noelle R. Thielman, Keyu Li, Lei Zheng, Kathleen Gabrielson, Phuoc T. Tran, Audrey Lafargue. Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model [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 1444.

Abstract 5573: Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma

Abstract Glutathione S-transferase omega 2 (GSTO2) is known as one of important regulators for the glutathione redox balance. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was completely lost in lung squamous cell carcinoma (LSCC) (Cancer Science, 2022). In this study, we examined the expression of GSTO2 in lung adenocarcinoma (LAC), another type of lung cancer, and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 139 patients who were diagnosed with LAC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. Fifty eight of 139 lung adenocarcinoma samples exhibited GSTO2 expression, while remaining 81 cases did not. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index < 400) smoking history (P=0.0027) and histological subtypes (lepidic 77%; acinar 44%; papillary 22%; micropapillary 16.7%; solid 9.6%; P<0.0001). There were no significant differences between the groups with respect to pN, pM, EGFR mutation, ALK fusion, and ROS-1 fusion. However, GSTO2 expression in LAC significantly associated with pT1 (P<0.0001), early stage (P<0.0001), and absence of PD-L1 expression (P=0.0027). Moreover, patients with GSTO2-negative LAC had a significantly lower disease-free survival rate than those with GSTO2-positve LAC (P=0.028). To examine whether GSTO2 expression affects PD-L1 expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell lines (A549 and PC-9). In both cell lines, mRNA level of PD-L1 was significantly reduced in GSTO2-transfectants. Because Jang RH et al reported that vimentin, reflecting to acquire mesenchymal traits in epithelial-mesenchymal transition (EMT), regulates the PD-L1 expression, we next examined whether GSTO2 overexpression affects the expression of EMT related molecules. There was no difference in a mRNA level of E-cadherin, while the mRNA expression of vimentin and SLUG, a transcriptional inducer of EMT, were significantly suppressed in GSTO2-transfected PC-9. In both cell lines, protein expression of vimentin was also significantly reduced in GSTO2-transfectants. The present study shed light on a novel function of GSTO2 as a PD-L1 regulator, which may contribute to prevention of tumor immune escape. Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Yuki I. Kawamura. Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma [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 5573.

Transcriptional and phenotypical alterations associated with a gradual benzo[a]pyrene-induced transition of human bronchial epithelial cells into mesenchymal-like cells

The role of benzo[a]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFβ1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression.

Reduced chemokine C-C motif ligand 1 expression may negatively regulate colorectal cancer progression at liver metastatic sites.

Colorectal cancer (CRC) liver metastasis, albeit a stage-IV disease, is completely curable by surgical resection in selected patients. In addressing the molecular basics of this phenomenon, differentially expressed genes at primary and liver metastatic sites were screened by RNA sequencing with the use of paraffin-embedded surgical specimens. Chemokine C-C motif ligand 1 (CCL1), a chemotactic factor for a ligand of the chemokine C-C motif receptor 8 (CCR8), was isolated as one of the differentially expressed genes. Histological analysis revealed that the number of CCL1-positive cells, mainly tumour associated macrophages (TAMs) located in the stroma of CRC, decreased significantly at liver metastatic sites, while the expression level of CCR8 on CRC remained unchanged. To explore the biological significance of the CCL1-CCR8 axis in CRC, CCR8-positive CRC cell line Colo320DM was used to assess the effect of the CCL1-CCR8 axis on major signalling pathways, epithelial mesenchymal transition induction and cell motility. Upon stimulation of recombinant CCL1 (rCCL1), phosphorylation of AKT was observed in Colo320DM cells; on the other hand, the corresponding significant increase in MMP-2 levels demonstrated by RT-qPCR was nullified by siRNA (siCCR8). In the scratch test, rCCL1 treatment significantly increased the motility of Colo320DM cells, which was similarly nullified by siCCR8. Thus, the activation of the CCL1-CCR8 axis is a positive regulator of CRC tumour progression. Reduced CCL1 expression of TAMs at liver metastatic sites may partly explain the unique slow tumour progression of CRC, thus providing for a grace period for radical resection of metastatic lesions.

Canonical DDR activation by EMT inducing agent 5-Fluorouracil is modulated by a cannabinoid based combinatorial approach via inducing autophagy and suppression of vimentin expression

Anastasis cascade including induction of Epithelial to Mesenchymal Transition (EMT), DNA repair, and stimulation of pro-survival mediators collectively exaggerate therapy resistance in cancer prognosis. The extensive implications of DNA-damaging agents are clinically proven futile for the rapid development of disease recurrence during treatment regime. Herein we report a glycosidic derivative of Δ9-tetrahydrocannabinol (THC-9-OG) abrogates sub-toxic doses of 5-Fluorouracil (5FU) induced EMT in colon cancer cells nullifying DNA repairing mechanism. Our in vitro and in vivo data strongly proclaims that THC-9-OG could not only abrogate 5FU mediated background EMT activation through stalling matrix degradation as well as murine 4T1 lung metastasis but also vigorously diminished Rad-51 repairing mediator along with stimulation of γ-H2AX foci formation. The combinatorial treatment (5FU + THC-9-OG) in Apc knockout colorectal carcinoma model conferred remission of the crypt progenitor phenotype which was prominently identified in 5FU treatment. Mechanistically, we demonstrated that 5FU plus THC-9-OG significantly attenuated major EMT inducer Vimentin via extensive ROS generation along with autophagy induction via LC3B I-II conversion and p62 degradation in a p-ATM dependent manner. Additionally, Cannabinoid receptor CB1 was responsible for abrogation of Vimentin since we found increase in the expression of γH2AX and decrease in vimentin expression in CB1 agonist (ACEA) plus 5FU treated cells. Nutshell, our results unveil a new direction of Cannabinoid based combinatorial approach to control background EMT along with robust enhancing of DNA damage potential of sub-toxic concentration of 5FU resulting immense inhibition of distant metastasis coupled with triggering cell death in vitro and in vivo.

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.

Long Non-coding RNA 02298 Promotes the Malignancy of HCC by Targeting the miR-28-5p/CCDC6 Pathway.

Hepatocellular carcinoma (HCC) is a malignancy characterized by a high fatality rate. Increasing evidence indicating that long non-coding RNAs (lncRNAs) play a regulatory role in hepatocellular carcinoma (HCC). Among them, the correlation between LINC02298 and HCC remains unknown. The expression and subcellular localization of LINC02298 in HCC tissues and cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, the correlation between the expression of LINC02298 and clinicopathological features of HCC patients was analyzed. The regulatory effects of LINC02298 in HCC were investigated using colony formation, cell count Kit-8(CCK8), Transwell, EDU, cell cycle and apoptosis analysis. In addition, the expression of EMT-related proteins were detected by western blotting. Dual-luciferase reporter, RT-qPCR and rescue assays were employed to validate the involvement of the LINC02298/miR-28-5p/CCDC6 axis in the progression of HCC. The up-regulation of LINC02298 was observed in hepatocellular carcinoma (HCC) tissues and cells, and it was found to be correlated with a negative prognosis in patients with HCC. Overexpression of LINC02298 enhanced the proliferation, migration, invasion, and induction of Epithelial-Mesenchymal Transition (EMT) while suppressing apoptosis in HCC cells. LINC02298 bind to miR-28-5p to regulate the expression of CCDC6. Inhibition of miR-28-5p saved the inhibitory effect of shLINC02298, and knockdown of CCDC6 also saved the inhibitory effect of miR-28-5p on HCC in vitro and in vivo. LINC02298 regulates the expression of CCDC6 by sponging of miR-28-5p, thereby facilitating the the malignancy and EMT of HCC.

NANOS1 restricts oral cancer cell motility and TGF-ß signaling

Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells.

Continuous genetic monitoring of transient mesenchymal gene activities in distal tubule and collecting duct epithelial cells during renal fibrosis.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-β, a major inducer of EMT.

The clustering status of detached gastric cancer cells inhibits anoikis-induced ferroptosis to promote metastatic colonization

Background and purposeFerroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. Its role in cancer metastasis remains unclear. In this study, we aimed to investigate the potential involvement of ferroptosis in gastric cancer (GC) metastasis.MethodsGC cells (AGS, MKN45, HGC27) were used to explore the role of ferroptosis in single and clustered cells with extracellular matrix (ECM) detachment in vitro. We overexpressed glutathione peroxidase 4 (GPX4) to inhibit ferroptosis and assessed the changes in cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Then tumor tissues from 54 GC patients with and without lymphatic metastasis were collected for immunohistochemical staining to investigate the expression of ferroptosis and EMT markers. Finally, Kaplan–Meier survival curves were used to investigate the relationship between overall survival and expression of GPX4 in 178 GC patients.ResultsDetached single cells had lower viability than adherent cells, but cell clustering improved their survival under matrix-detached conditions. Detached single cells exhibited an induction of iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of ACSL4, TFRC and HO-1, increased iron levels, and changes in mitochondrial morphology. Opposite effects were observed in detached clustered cells, including the upregulation of the ferroptosis suppressors GPX4 and SLC7A11. Overexpression of GPX4 inhibited ferroptosis and promoted GC cell proliferation, migration, invasion, and EMT. Immunohistochemical analysis of tumor tissues from GC patients indicated that lymphatic metastasis was associated with higher potential for ferroptosis inhibition and EMT induction. Finally, Kaplan–Meier survival curves demonstrated a significant decrease in overall survival among GC patients with high GPX4 expression.ConclusionsOur study provides the first evidence that inhibition of ferroptosis is a crucial mechanism promoting GC metastasis. GPX4 may be a valuable prognostic factor for GC patients. These findings suggest that targeting ferroptosis inhibition may be a promising strategy for GC patients with metastatic potential.Trial registration The ethical approval code of this study in Institutional Review Board of Peking Union Medical College Hospital is No: K1447.