Epithelial-mesenchymal Transition Phenotype Research Articles

Abstract PO2-24-02: Molecular characterization of primary tumors associated with various CTCs’ subpopulation

Abstract Background: Circulating tumor cells (CTCs) are prognostic in primary breast cancer (BC) patients and represent a heterogeneous population of cells with different phenotypes and biological value. However, molecular characterization of primary tumors associated with various CTCs’ subpopulation is lacking. This study aimed to identify expression of genes associated with the presence of different CTCs subpopulations in primary BC patients using a comprehensive genomics approach. Methods: This prospective ongoing study included 312 patients (pts) with non-metastatic BC enrolled from March 2012 to February 2015. CTCs were detected on day -1 to 0 before surgery. Isolated peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSepTM kit negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45-) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC of patients were compared to those of CD45- PBMC of 60 healthy donors. Formalin-fixed paraffin-embedded tumor tissue was subject for gene expression analysis performed by HTG EdgeSeq Oncology Biomarker panel (HTG Molecular Diagnostics, Inc., Tucson, USA). Results: CTCs with epithelial phenotype (CTC_EP) were detected in 30 (9.6%) pts, CTC with EMT (epithelial-to-mesenchymal transition) were detected in 54 (17.3%), while blood of 79 (25.3%) pts exhibit at least one type of CTC. Patients with detectable CTC in peripheral blood had inferior disease-free survival compared to patients without detectable CTC (HR = 0.62, 95% CI 0.37-1.06, P=0.048). There was no association between epithelial CTCs and analysed patients/tumour variables. Genes expression analysis identified 7 differentially expressed genes in patients with the CTC_EP phenotype (CHGA, MESP1, COL2A1, MT2A, COMP, MMP7 and ORM2), 200 genes associated with CTC_EMT phenotype (top 10 genes included CHGA, CECR6, SMAD2, HUS1, FGF10, HNF1A, SEC61G, CDK4, CD44 and F3). Analysis identified that low expression of CHGA and MMP7 was consistently associated with any CTC subpopulations. Conclusions: In this study for the first-time revealed gene expressions associated with various CTC subpopulations. We suppose that these genes could represent potential therapeutic target aimed to inhibit metastatic cascade in breast cancer. Citation Format: Michal Mego, Gabriel Minarik, Milan Hucko, Katarina Kalavska, Marian Karaba, Juraj Benca, Tatiana Sedlackova, Lucia Kucerova, Jozef Mardiak, Lubos Klucar, Zuzana Cierna. Molecular characterization of primary tumors associated with various CTCs’ subpopulation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-02.

TSPYL1 as a Critical Regulator of TGFβ Signaling through Repression of TGFBR1 and TSPYL2.

Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFβ causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFβ signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFβ signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFβ to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFβ signaling is also unraveled.

TGF-β downstream of Smad3 and MAPK signaling antagonistically regulate the viability and partial epithelial-mesenchymal transition of liver progenitor cells.

Liver progenitor cells (LPCs) are a subpopulation of cells that contribute to liver regeneration, fibrosis and liver cancer initiation under different circumstances. By performing adenoviral-mediated transfection, CCK-8 analyses, F-actin staining, transwell analyses, luciferase reporter analyses and Western blotting, we observed that TGF-β promoted cytostasis and partial epithelial-mesenchymal transition (EMT) in LPCs. In addition, we confirmed that TGF-β activated the Smad and MAPK pathways, including the Erk, JNK and p38 MAPK signaling pathways, and revealed that TGFβ-Smad signaling induced growth inhibition and partial EMT, whereas TGFβ-MAPK signaling had the opposite effects on LPCs. We further found that the activity of Smad and MAPK signaling downstream of TGF-β was mutually restricted in LPCs. Mechanistically, we found that TGF-β activated Smad signaling through serine phosphorylation of both the C-terminal and linker regions of Smad2 and 3 in LPCs. Additionally, TGFβ-MAPK signaling inhibited the phosphorylation of Smad3 but not Smad2 at the C-terminus, and it reinforced the linker phosphorylation of Smad3 at T179 and S213. We then found that overexpression of mutated Smad3 at linker phosphorylation sites intensifies TGF-β-induced cytostasis and EMT, mimicking the effects of MAPK inhibition in LPCs, whereas mutation of Smad3 at the C-terminus caused LPCs to blunt TGF-β-induced cytostasis and partial EMT. These results suggested that TGF-β downstream of Smad3 and MAPK signaling were mutually antagonistic in regulating the viability and partial EMT of LPCs. This antagonism may help LPCs overcome the cytostatic effect of TGF-β under fibrotic conditions and maintain partial EMT and progenitor phenotypes.

SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1–involved EMT in hepatocellular carcinoma

BackgroundThe protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression.MethodsThe modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient’s overall survival versus AnxA6 expression level was evaluated by the Kaplan–Meier method.ResultsLys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients.ConclusionsAnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

Co-Expression Network Analysis Unveiled lncRNA-mRNA Links Correlated to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance and/or Intermediate Epithelial-to-Mesenchymal Transition Phenotypes in a Human Non-Small Cell Lung Cancer Cellular Model System.

We investigated mRNA-lncRNA co-expression patterns in a cellular model system of non-small cell lung cancer (NSCLC) sensitive and resistant to the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib/gefitinib. The aim of this study was to unveil insights into the complex mechanisms of NSCLC targeted therapy resistance and epithelial-to-mesenchymal transition (EMT). Genome-wide RNA expression was quantified for weighted gene co-expression network analysis (WGCNA) to correlate the expression levels of mRNAs and lncRNAs. Functional enrichment analysis and identification of lncRNAs were conducted on modules associated with the EGFR-TKI response and/or intermediate EMT phenotypes. We constructed lncRNA-mRNA co-expression networks and identified key modules and their enriched biological functions. Processes enriched in the selected modules included RHO (A, B, C) GTPase and regulatory signaling pathways, apoptosis, inflammatory and interleukin signaling pathways, cell adhesion, cell migration, cell and extracellular matrix organization, metabolism, and lipid metabolism. Interestingly, several lncRNAs, already shown to be dysregulated in cancer, are connected to a small number of mRNAs, and several lncRNAs are interlinked with each other in the co-expression network.

Abstract 6247: Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) and linitis-plastica type gastric cancer (STAD) are the most representative refractory cancers even in the era of molecularly targeted therapy and immunotherapy. Extensive characterization of these tumors, especially regarding the genomic and epigenomic characteristics of pure cancer cells, has been hampered by the high stromal content in the primary tumor samples. Here we took advantage of cancer cells purified from malignant ascites and established corresponding cell lines to overcome the limitations associated with the analysis and to discover their vulnerabilities. Methods: We developed cell lines from malignant ascitic fluid samples obtained from 60 patients with PDAC and 59 patients with linitis-plastica type STAD. We performed comprehensive multi-omic analyses of these cell lines using long-read whole-genome sequencing (WGS) for complex somatic genomic alterations and for methylation aberration. We also conducted long-read RNA sequencing (iso-seq) for full-length isoform exploration, and chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for enhancer analysis. Complementary short-read whole-genome sequencing and RNA sequencing were also performed. Results: Comprehensive transcriptome analyses identified a cluster with high epithelial-mesenchymal transition (EMT) characteristics, consisting of the majority of the PDAC cell lines and a half of the STAD cell lines. ChIP-seq analysis against H3K27ac revealed the distinct transcriptional circuit that generated EMT cluster-specific super-enhancers governed by SMAD3, independent of cancer type. Most of the transcriptional activity was controlled by the gene methylation. We also identified the differential expression levels of the splicing factors between the EMT-activated and the non-activated groups. Comprehensive isoform analysis by iso-seq revealed that the isoform usage was completely different between the EMT group and the other, especially in genes of the actomyosin pathway which confer characteristic features of the EMT cluster in the cell morphology and cell motility. Using the CHM13 reference genome, long-read WGS analyses revealed the recurrent structural alterations specific to the EMT group such as those involving CDKN2A. We also identified PDAC-specific structural alterations. Conclusion: We identified the distinct molecular characteristics of the EMT-activated group in PDAC and linitis-plastica type STAD which may confer the therapeutic resistance. Our findings provide an interesting insight for the further innovation of the treatment strategy for these intractable cancers. Citation Format: Yosuke Tanaka, Hiroyuki Mano. Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing [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 6247.

Abstract 277: Cancer-associated fibroblasts, educated by tumor cells, differentially facilitate cancer cell invasion in cell-line based human gastric tumoroids

Abstract Purposes: Gastric cancer can be categorized into two subtypes: intestinal and diffuse. The latter is notorious for frequent peritoneal metastasis, resulting in a poorer prognosis. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play crucial roles in tumor invasion and metastasis. However, little is known about whether CAFs are associated with the differential progression between the two subtypes of gastric tumors. This study investigated whether differential cancer invasion may be attributed to tumor-specific CAF activation, using a cell-line-based human gastric tumoroid model. Methods: Heterotypic tumor spheroids (TS) were prepared with human gastric cancer lines of intestinal (MKN28, MKN74) and diffuse type (MKN45, KATO III) mixed with human gastric fibroblasts (HGFs) using a minipillar-array chip previously developed. Invasion phenotype and expression of epithelial-mesenchymal transition (EMT)-related proteins were evaluated in heterotypic TSs. mRNA expression in HGFs isolated from heterotypic TSs was analyzed and compared between the two subtypes. Results: While both heterotypic TSs displayed increased invasion by HGFs, the diffuse type TS showed a higher level of invasion with individual or amoeboid migration and loose binding of cancer cells with HGF. In contrast, the intestinal type TS showed collective migration and tight binding between cancer cells and HGFs. HGFs in each heterotypic TS exhibited differential morphology, matrix remodeling patterns, and gene expression in a tumor subtype-specific manner. Compared to normal HGFs, HGFs isolated from diffuse heterotypic TS showed enriched expression of cytoskeleton-associated genes and several integrins, whereas those of intestinal heterotypic TS exhibited increased expression levels of inflammatory cytokines. Conclusions: This study established tumoroid models for human gastric cancer subtypes, intestinal and diffuse, using heterotypic TS culture. Our results suggest that the subtype-specific invasion ability of tumor cells is driven by CAFs that are differentially educated by tumor cells in the tumor microenvironment. Our model not only advances our understanding of CAFs' role in tumor progression but also offers an in vitro model for assessing potential therapeutic targets and drug candidates for human gastric cancers. Citation Format: Hyun Ju Hwang, Saet-Byeol Lee, Hyo-Jeong Kuh. Cancer-associated fibroblasts, educated by tumor cells, differentially facilitate cancer cell invasion in cell-line based human gastric tumoroids [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 277.

Abstract 4219: Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing

Abstract Several previous studies have highlighted the significance of cell-to-cell communication, or crosstalk, between tumor cells and non-tumorigenic cells in the context of cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have the potential to alter the phenotypes and behaviors of normal cells. The use of 2D in-vitro cultures has been limited due to their inability to accurately replicate the intricate in-vivo TME. Conditioned medium (CM) derived from cultured cancer cells contains secreted factors that may influence the phenotype and functionality of normal cells. In our investigation, the exposure of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells to conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in a modified phenotype with enhanced cell viability. Treatment with 4T1CM led to the upregulation of genes, including α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF), in NIH3T3 and RAW 264.7 cells compared to their respective control cells. Additionally, NIH3T3 cells treated with 4T1CM exhibited an epithelial-mesenchymal transition (EMT) phenotype, as indicated by the regulation of EMT markers such as E-cadherin, β-catenin, N-cadherin, and Vimentin. Notably, RAW 264.7 cells treated with 4T1CM showed an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1), suggesting a propensity for an inhibitory immune response. Moreover, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers, including sex determining region Y-box 2 and Aldehyde dehydrogenase. In summary, our study highlights the potential role of 4T1CM in transforming normal NIH3T3 and RAW 264.7 cells into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Ongoing proteomics analysis aims to map differentially expressed proteins potentially regulating the 4T1CM-induced transformation. To further investigate, a series of experiments using mono- and co-cultures of these cells are underway, specifically targeting the transformed macrophages and fibroblasts with the aim of either re-educating or eliminating them. This treatment involves drug combinations that specifically target the identified signaling pathway intermediates, offering novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Amirsalar Mansouri, Jiri Adamec, Joseph Francis. Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing [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 4219.

Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

Abstract Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC. Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice. Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo. Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment. Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [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 6992.

Abstract 2884: Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC

Abstract Receptor tyrosine kinases (RTK) and integrins coalesce at focal adhesions. These cell membrane areas serve as huge signaling hubs critically contributing to central aspects of cancer cell survival and therapy resistance. Despite the fact that these receptors mutually interact, co-dependencies between these receptors and associated therapeutically exploitable vulnerabilities remain largely unclear in HPV-negative head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin in up to 20 different 3D matrix grown HNSCC cell models. RNA sequencing and protein-based biochemical assays were performed for molecular and pathway characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts to show clinical relevance. Our observations demonstrate that fibroblast growth factor receptors (FGFR 1-4) present with the strongest cytotoxic and radiosensitizing potential, both as monotherapy and in combination with β1 integrin inhibition, surpassing the efficacy of the other investigated RTK. Pharmacological pan-FGFR inhibition elicited a variable response spectrum ranging from cytotoxicity/radiochemosensitization to resistance/radioprotection. Transcriptomic characterization revealed an association of these contrasting responses to FGFR inhibition with a mesenchymal-to-epithelial transition (MET) for sensitive cell models and a partial epithelial-to-mesenchymal transition (EMT) for resistant cell models. Accordingly, deactivation of EMT associated kinases like EGFR, PKC and PAK proved effective in diminishing the adaptive FGFR-driven resistance. Importantly, the translation of the adaptive resistance profiles to HNSCC patient cohorts showed its prognostic value and provided conclusive validation of the presence of EMT-related vulnerabilities that can be strategically used for therapeutic intervention. In conclusion, our study demonstrates that pan-FGFR inhibition induces both a highly beneficial radiochemosensitizing and a detrimental radioprotective effect in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically. Citation Format: Felix Broghammer, Mahesh Gouda, Irina Korovina, Cornelia Brunner, Robert P. Coppes, Olivier Gires, Michael Seifert, Nils Cordes. Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC [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 2884.

Abstract 1740: Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion

Abstract Metastatic potential of basal-like breast cancers typically is initiated by genetic alterations that lead to a process known as epithelial-mesenchymal transition (EMT). However, much is currently not understood regarding the role of epigenetic modifications that lead to invasive characteristics and EMT phenotype of metastatic breast cancers. Based on the previous notion connecting epigenetic changes to breast cancer metastasis, we performed DIA-based mass spectrometry of isolated histones from an isogenic panel of MCF10A breast cell lines where the tumor suppressor genes TP53 and PTEN were silenced and induced EMT. With this approach, we determined which histone modifications were differentially enriched in the non-EMT and EMT-induced MCF10A cell lines. From approximately 72 histone modifications identified and annotated from our mass spectrometry results, we were able to identify 5 histone events that were differentially enriched in our MCF10A cell line panel. Two events of note were histone H3 lysine-14 acetylation (H3K14ac) significantly increasing and histone H4 arginine 55 dimethylation (H4R55me2) significantly decreasing in our EMT-transformed MCF10A p53-/PTEN- cell lines when compared to the parental, non-tumorigenic MCF10A cell line, showing these events are differentially affected during the EMT process in breast cancer cells. Furthermore, significant arginine demethylation of H4R55me2 & H3.1R83me in the EMT-transformed MCF10A p53-/PTEN- cell lines corresponded with JMJD6, an established histone arginine demethylase, being overexpressed in basal-like breast cancer cell lines as well as basal-like breast cancer patients from The Cancer Genome Atlas (TCGA) and METABRIC datasets. Based on histone proteomic profiling of our isogenic cell line model, the loss of specific histone arginine methylation events corresponding with JMJD6 overexpression could highlight the potential for a targetable epigenetic mechanism in breast cancer metastasis. Citation Format: Charlotte B. McGuinness, Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion [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 1740.

Abstract 6801: Cellular senescence involves spatial evolution in colorectal cancer

Abstract Cancer evolution is a continuous process involving cellular changes that promote progression and metastasis. Recent studies demonstrate that cancer evolution is spatially associated: a phenomenon called “the spatial evolution of cancer.” In our data, colorectal cancer cells gradually evolve from an adenoid to a collective invasion morphology, ultimately developing a partial epithelial-mesenchymal transition phenotype as they form an invasive front. Trajectory analysis showed that cancer cells at the invasive front of a tumor exhibit a totally different gene expression pattern from those at the center, and that cellular senescence plays a crucial role in this transition. Epigenetic changes in the promoter/exon1 region of the CDKN2A gene caused by increased reactive oxygen species level are crucial for expression of p16INK4A and induction of senescence in the cancer cells. Two types of senescent tumor cells (type I and type II) were observed, and they played different roles in cancer progression due to different gene expression. Type II senescent tumor cells (p16INK4A+/LAMC2+/MMP7+), a final evolved form of cancer cells, are strongly associated with local invasion and lymph node metastasis of colorectal cancer and worsen the prognosis for patients. Citation Format: Tae Jun Park, Seok Yun Kang, Jang Hee Kim, Hyun Woo Lee, Yong Won Choi, Soon Sang Park, Young-Kyoung Lee. Cellular senescence involves spatial evolution in colorectal 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 6801.

LINC00540 promotes sorafenib resistance and functions as a ceRNA for miR-4677-3p to regulate AKR1C2 in hepatocellular carcinoma

Sorafenib resistance is one of the main causes of poor prognosis in patients with advanced hepatocellular carcinoma (HCC). Long noncoding RNAs (lncRNAs) function as suppressors or oncogenic factors during tumor progression and drug resistance. Here, to identify therapeutic targets for HCC, the biological mechanisms of abnormally expressed lncRNAs were examined in sorafenib-resistant HCC cells. Specifically, we established sorafenib-resistant HCC cell lines (Huh7-S and SMMC7721-S), which displayed an epithelial-mesenchymal transition (EMT) phenotype. Transcriptome sequencing (RNA-Seq) was performed to established differential lncRNA expression profiles for sorafenib-resistant cells. Through this analysis, we identified LINC00540 as significantly up-regulated in sorafenib-resistant cells and a candidate lncRNA for further mechanistic investigation. Functionally, LINC00540 knockdown promoted sorafenib sensitivity and suppressed migration, invasion, EMT and the activation of PI3K/AKT signaling pathway in sorafenib-resistant HCC cells, whereas overexpression of LINC00540 resulted in the opposite effects in parental cells. LINC00540 functions as a competing endogenous RNA (ceRNA) by competitively binding to miR-4677-3p , thereby promoting AKR1C2 expression. This is the first study that demonstrates a role for LINC00540 in enhancing sorafenib resistance, migration and invasion of HCC cells through the LINC00540/miR-4677-3p/AKR1C2 axis, suggesting that LINC00540 may represent a potential therapeutic target and prognosis biomarker for HCC.

TGIF2 is a potential biomarker for diagnosis and prognosis of glioma.

TGFB-induced factor homeobox 2 (TGIF2), a member of the Three-Amino-acid-Loop-Extension (TALE) superfamily, has been implicated in various malignant tumors. However, its prognostic significance in glioma, impact on tumor immune infiltration, and underlying mechanisms in glioma development remain elusive. The expression of TGIF2 in various human normal tissues, normal brain tissues, and gliomas was investigated using HPA, TCGA, GTEx, and GEO databases. The study employed several approaches, including Kaplan-Meier analysis, ROC analysis, logistic regression, Cox regression, GO analysis, KEGG analysis, and GSEA, to explore the relationship between TGIF2 expression and clinicopathologic features, prognostic value, and potential biological functions in glioma patients. The impact of TGIF2 on tumor immune infiltration was assessed through Estimate, ssGSEA, and Spearman analysis. Genes coexpressed with TGIF2 were identified, and the protein-protein interaction (PPI) network of these coexpressed genes were constructed using the STRING database and Cytoscape software. Hub genes were identified using CytoHubba plugin, and their clinical predictive value was explored. Furthermore, in vitro experiments were performed by knocking down and knocking out TGIF2 using siRNA and CRISPR/Cas9 gene editing, and the role of TGIF2 in glioma cell invasion and migration was analyzed using transwell assay, scratch wound-healing assay, RT-qPCR, and Western blot. TGIF2 mRNA was found to be upregulated in 21 cancers, including glioma. High expression of TGIF2 was associated with malignant phenotypes and poor prognosis in glioma patients, indicating its potential as an independent prognostic factor. Furthermore, elevated TGIF2 expression positively correlated with cell cycle regulation, DNA synthesis and repair, extracellular matrix (ECM) components, immune response, and several signaling pathways that promote tumor progression. TGIF2 showed correlations with Th2 cells, macrophages, and various immunoregulatory genes. The hub genes coexpressed with TGIF2 demonstrated significant predictive value. Additionally, in vitro experiments revealed that knockdown and knockout of TGIF2 inhibited glioma cell invasion, migration and suppressed the epithelial-mesenchymal transition (EMT) phenotype. TGIF2 emerges as a potential biomarker for glioma, possibly linked to tumor immune infiltration and EMT.

Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome

BackgroundFocal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC).MethodsThe cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts.ResultsFibroblast growth factor receptor (FGFR 1–4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with β1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions.ConclusionsThis study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

Targeting Senescent Alveolar Epithelial Cells Using Engineered Mesenchymal Stem Cell-Derived Extracellular Vesicles To Treat Pulmonary Fibrosis.

Type 2 alveolar epithelial cell (AEC2) senescence is crucial to the pathogenesis of pulmonary fibrosis (PF). The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme cluster of differentiation 38 (CD38) is a marker of senescent cells and is highly expressed in AEC2s of patients with PF, thus rendering it a potential treatment target. Umbilical cord mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) have emerged as a cell-free treatment with clinical application prospects in antiaging and antifibrosis treatments. Herein, we constructed CD38 antigen receptor membrane-modified MSC-EVs (CD38-ARM-MSC-EVs) by transfecting MSCs with a lentivirus loaded with a CD38 antigen receptor-CD8 transmembrane fragment fusion plasmid to target AEC2s and alleviate PF. Compared with MSC-EVs, the CD38-ARM-MSC-EVs engineered in this study showed a higher expression of the CD38 antigen receptor and antifibrotic miRNAs and targeted senescent AEC2s cells highly expressing CD38 in vitro and in naturally aged mouse models after intraperitoneal administration. CD38-ARM-MSC-EVs effectively restored the NAD+ levels, reversed the epithelial-mesenchymal transition phenotype, and rejuvenated senescent A549 cells in vitro, thereby mitigating multiple age-associated phenotypes and alleviating PF in aged mice. Thus, this study provides a technology to engineer MSC-EVs and support our CD38-ARM-MSC-EVs to be developed as promising agents with high clinical potential against PF.

Baicalin induces ferroptosis in oral squamous cell carcinoma by suppressing the activity of FTH1.

This study investigated the role of the ferroptosis-related gene FTH1 in oral squamous cell carcinoma (OSCC) and evaluated the therapeutic potential of baicalin in OSCC cell treatment. A prognostic model was established by bioinformatic analysis, consisting of 12 ferroptosis related genes (FRGs), and FTH1 was selected as the most significantly up-regulated FRGs. The clinical correlation of FTH1 in OSCC samples was evaluated by both immunohistochemical and bioinformatic characterizations. The effects of FTH1 on migration, invasion, epithelial-mesenchymal transition (EMT) and proliferation were determined by wound healing assays, transwell assays, western blotting and 5'-ethynl 2'-deoxyuridine proliferation assays, respectively. The effects of FTH1 on ferroptosis were tested via ferroptosis markers and Mito Tracker staining. In addition, the therapeutic effects of baicalin on OSCC cells were confirmed using EMT, migration, invasion, proliferation and ferroptosis assays. The 12 FRGs werepredictive of theprognosis for OSCC patients, and FTH1 expression was identified as significantly up-regulated in OSCC samples, which was highly associated with survival, immune cell infiltration and drug sensitivity. Moreover, knocking down FTH1 inhibited cell proliferation, EMT and invasive phenotypes, but induced ferroptosis in OSCC cells (Cal27 and SCC25). Furthermore, baicalin directly suppressed expression of FTH1 in OSCC cells, and effectively promoted ferroptosis and inhibited the proliferation as well as EMT by directly targeting FTH1. This study has demonstrated that FTH1 is a therapeutic target for OSCC treatment, and has provided evidence that baicalin offers a promising alternative for OSCC treatment.

The TGFβ2-Snail1-miRNATGFβ2 Circuitry is Critical for the Development of Aggressive Functions in Breast Cancer.

There have been contradictory reports on the biological role of transforming growth factor-βs (TGFβs) in breast cancer (BC), especially with regard to their ability to promote epithelial-mesenchymal transition (EMT). Here, we show that TGFβ2 is preferentially expressed in mesenchymal-like BCs and maintains the EMT phenotype, correlating with cancer stem cell-like characteristics, growth, metastasis and chemo-resistance and predicting worse clinical outcomes. However, this is only true in ERα- BC. In ERα+ luminal-type BC, estrogen receptor interacts with p-Smads to block TGFβ signalling. Furthermore, we also identify a microRNAs (miRNAs) signature (miRNAsTGFβ2 ) that is weakened in TGFβ2-overexpressing BC cells. We discover that TGFβ2-Snail1 recruits enhancer of zeste homolog-2 to convert miRNAsTGFβ2 promoters from an active to repressive chromatin configuration and then repress miRNAsTGFβ2 transcription, forming a negative feedback loop. On the other hand, miRNAsTGFβ2 overexpression reverses the mesenchymal-like traits in agreement with the inhibition of TGFβ2-Snail1 signalling in BC cells. These findings clarify the roles of TGFβ2 in BC and suggest novel therapeutic strategies based on the TGFβ2-Snail1-miRNAsTGFβ2 loop for a subset type of human BCs.

Inhibition of proprotein convertases activity results in repressed stemness and invasiveness of cancer stem cells in gastric cancer.

Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs' inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC.

AEBP1 promotes papillary thyroid cancer progression by activating BMP4 signaling

Papillary thyroid cancer (PTC) is the most prevalent endocrine cancer worldwide. Approximately 30 % of PTC patients will progress into the advanced or metastatic stage and have a relatively poor prognosis. It is well known that epithelial-mesenchymal transition (EMT) plays a pivotal role in thyroid cancer metastasis, resistance to therapy, and recurrence. Clarifying the molecular mechanisms of EMT in PTC progression will help develop the targeted therapy of PTC. The aberrant expression of some transcription factors (TFs) participated in many pathological processes of cancers including EMT. In this study, by performing bioinformatics analysis, adipocyte enhancer-binding protein 1 (AEBP1) was screened as a pivotal TF that promoted EMT and tumor progression in PTC. In vitro experiments indicated that knockout of AEBP1 can inhibit the growth and invasion of PTC cells and reduce the expression of EMT markers including N-cadherin, TWIST1, and ZEB2. In the xenograft model, knockout of AEBP1 inhibited the growth and lung metastasis of PTC cells. By performing RNA-sequencing, dual-luciferase reporter assay, and chromatin immunoprecipitation assay, Bone morphogenetic protein 4 (BMP4) was identified as a downstream target of AEBP1. Over-expression of BMP4 can rescue the inhibitory effects of AEBP1 knockout on the growth, invasion, and EMT phenotype of PTC cells. In conclusion, these findings demonstrated that AEBP1 plays a critical role in PTC progression by regulating BMP4 expression and the AEBP1-BMP4 axis may present novel therapeutic targets for PTC treatment.