EMT-inducing Transcription Factors 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.

Abstract LB225: Regulation of breast cancer stem cell identity by PLK1 and FOXC2

Abstract One of the main drivers for the drastic reduction in survival rates caused by metastatic breast cancer is attributed to Epithelial-to-Mesenchymal Transition (EMT). During embryonic development and wound healing, EMT plays a vital role and during cancer pathogenesis, cancer cells reactivate this program to gain plasticity, migration, and invasion promoting metastasis. In addition, EMT also plays a crucial role in generating a subpopulation of highly resilient tumor cells known as cancer stem cells (CSCs). Moreover, following conventional treatment, residual tumor cells that remain after treatment tend to exhibit CSC properties. EMT-induced cancer stem cells exhibit heightened plasticity and stemness, significantly fueling the metastatic spread of cancer and enhancing the endurance and adaptability throughout the process of metastasis. Thus, understanding the molecular intricacies governing stem cell properties holds immense potential for developing targeted therapies to target metastasis and the development of resistance to treatments. To further elucidate EMT-induced cancer stem cell generation, our lab has demonstrated that the Polo-like kinase 1 (PLK1), an important G2/M regulator, plays an important role in the generation of CSCs through Forkhead box protein C2 (FOXC2). Most importantly, we have shown that the FOXC2 transcription factor, known to be crucial in embryonic development, now has emerging roles in metastatic cancer progression. FOXC2 is positioned at the center of the complex network of signal transduction pathways and functions downstream of most EMT-inducing transcription factors. FOXC2 is necessary for the acquisition and maintenance of stem cell property. In addition, we found that phosphorylation of FOXC2 by PLK1 stabilizes, and inhibition of PLK1 causes a decrease in FOXC2 and stemness in cancer cells. This led us to hypothesize that EMT-induced CSC generation is driven by the PLK1-FOXC2 axis. By expressing phosphomimetic and non-phosphorylatable FOXC2 mutants in breast cancer cell lines, we show that PLK1 phosphorylation is necessary for gaining stemness and an increase in symmetric self-renewal type of stem cell division. Evidence will be presented as to how we are targeting the PLK1-FOXC2 axis to make tumors sensitive to standard-of-care treatments. Citation Format: Joanna Joyce Maddela, Maria Castaneda, Petra den Hollander, Nick Kuburich, Sendurai Mani. Regulation of breast cancer stem cell identity by PLK1 and FOXC2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB225.

Abstract 4265: ZEB1-regulated hyaluronan network in the lung tumor microenvironment

Abstract In the tumor microenvironment, hyaluronan surrounds neighboring cells as one of the major components of the extracellular matrix and stimulates the epithelial-mesenchymal transition (EMT) of various epithelial cancer cells. However, the mechanism of how the hyaluronic network is formed by cancer cells and regulates cancer progression and metastasis has not been known in detail. Therefore, we investigated the role of the hyaluronan network built-up by cancer cells in regulating cancer progression and metastasis in the tumor microenvironment. Our study demonstrated that ZEB1, an EMT-inducing transcription factor, reconstructs the hyaluronan network to induce the expression of ITIH2, a hyaluronan-binding protein, and hyaluronic acid synthase-2 (HAS2), ZEB1 was also shown to control the isoform switching of CD44, a hyaluronan receptor. ITIH2 knockdown and HAS inhibition reduced the formation of hyaluronan cables and inhibited cancer cell migration and invasion. In addition, the treatment of a specific ITIH2 inhibitor identified through deep learning-based screening suppressed the formation of hyaluronan cables, cell migration, and metastasis. These findings suggest that targeting the hyaluronan network could be a novel strategy for suppressing lung cancer progression and metastasis in the tumor microenvironment. Citation Format: Sieun Lee, Jihye Park, Eun Ju Kim, Seongran Cho, Sungsoo Park, Jonathan M Kurie, Young-Ho Ahn. ZEB1-regulated hyaluronan network in the lung tumor microenvironment [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 4265.

TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT.

DOCK1 has been reported to be involved in tumor progression and resistance. 1-(2-(30-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-2-oxoethyl)-5-pyrrolidinylsulfonyl2(1H)- pyridone (TBOPP) is a selective DOCK1 inhibitor; however, the role and molecular mechanisms of DOCK1 and its inhibition in breast cancer (BC) resistance remain poorly understood. This study aims toinvestigate the underlying mechanisms of DOCK1 in BC resistance. DOCK1 or Twist siRNA and Twist plasmid were used to explore the function of DOCK1 in vitro experiments. A mouse xenograft model was used for in vivo experiments. In the present study, we demonstrated that DOCK1 siRNA promoted cisplatin sensitivity in BC cells. Moreover, TBOPP also enhances the therapeutic effect of cisplatin both in vitro and in vivo. Mechanistically, DOCK1 siRNA inhibited EMT. Twist 1 is one of the EMT-inducing transcription factors and is known to induce EMT. To further reveal the effect of DOCK in BC cells, we co-transfected with DOCK1 and Twist1 siRNA to BC cells and found that co-transfection with DOCK1 and Twist siRNA could not further enhance the cisplatin sensitivity of BC cells. Moreover, DOCK1 siRNA failed to reverse the effect of Twist 1 up-regulation. Taken together, these results demonstrate that DOCK1 may function as a potential therapeutic target in BC and that combining cisplatin with TBOPP may provide a promising therapeutic strategy for cisplatin-resistant BC patients.

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.

Crosstalk between cancer cell plasticity and immune microenvironment in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly aggressive tumor of the biliary tree characterized by an intense desmoplastic tumor microenvironment (TME). To date, treatment of CCA remains challenging; tumor resection is the only curative treatment with a high recurrence probability. Besides resection, therapeutic options have moved forward with the advent of immunotherapies, but these remain limited and low effective. Our knowledge about the cellular interplays in CCA is still fragmentary. An area is currently emerging regarding the potential role of cancer cell plasticity in the genesis of an immunosuppressive microenvironment. The cancer cells’ ability to acquire stemness properties and to disseminate through an epithelial-mesenchymal transition (EMT) shape a tumor immune microenvironment that supports cancer progression by attracting immunosuppressive cells including myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), M2 macrophages, and by increasing the expression of inhibitory immune checkpoints such as PD-1/PD-L-1. EMT-inducing transcription factors (EMT-TF) have recently emerged as regulators of tumor immunity by creating an immunosuppressive microenvironment. This review delves into the molecular mechanisms underlying the existing links between EMT/stemness and tumor immune microenvironment, as well as the last discoveries in CCA.

Nidogen-1/NID1 Function and Regulation during Progression and Metastasis of Colorectal Cancer.

We have previously shown that the extracellular matrix and basement membrane protein Nidogen1 (NID1) is secreted by more malignant, mesenchymal-like CRC cells and induces the epithelial-mesenchymal transition (EMT) and promotes the migration and invasion of less malignant, epithelial-like CRC cells. Here, we performed a comprehensive bioinformatics analysis of multiple datasets derived from CRC patients and showed that elevated expression of NID1 and the genes ITGA3, ITGB1, and ITGAV, which encode NID1 receptors, is associated with poor prognosis and advanced tumor stage. Accordingly, the expression of NID1, ITGA3, ITGB1, and ITGAV was associated with an EMT signature, which included SNAIL/SNAI1, an EMT-inducing transcription factor. In CRC cells, ectopic SNAIL expression induced NID1 and SNAIL occupancy was detected at an E-box upstream of the NID1 transcription start site. Therefore, NID1 represents a direct target of SNAIL. Ectopic expression of NID1 or treatment with NID1-containing medium endowed non-metastatic CRC cells with the capacity to form lung metastases after xenotransplantation into mice. Suppression of the NID1 receptor ITGAV decreased cell viability, particularly in CMS/consensus molecular subtype 4 CRC cells. Taken together, our results show that NID1 is a direct target of EMT-TF SNAIL and is associated with and promotes CRC progression and metastasis. Furthermore, the NID1 receptor ITGAV represents a candidate therapeutic target in CMS4 colorectal tumors.

(083) EMT Induced Transcription Factors Direct Leptin-LEPR-DHH Signaling to Modulate Leydig Stem Cells Differentiation in Testis

Abstract Introduction The primary source of testosterone (T) in men are Leydig cells (LCs) and their dysfunction can lead to T deficiency. Testicular microenvironment (TME) which is comparised of cells such as Sertoli and Peritubular myoid cells releases the paracrine factors to influence the growth and differentiation of LCs. Leptin is a 16-kDa protein produced primarily by adipose tissue and exerts a significant influence on hormonal regulation. In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cell (LSCs) differentiation and subsequent T production under the regulation of the desert hedgehog signalling pathway. We also showed that this regulation is inverse concentration dependent such as Leptin increases LSC differentiation at low doses and decrease it at higher doses. However, an in-depth understanding of mechanisms that derive the Leptin secretion and decide the fate of LCs are still unclear and is the focus of the present study. Objective To study the mechanisms that derive the Leptin secretion and decide the fate of LCs. Methods A total of 18 men with testicular failure underwent testis biopsies. The testicular cells were characterized using Flow cytometry, Immunostaining and RNA sequencing then the cells were also treated with Leptin in a dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) manner. For the functional studies, UCSC genome browser was used to study the transcription factor genes (TFs) binding the leptin receptor (LEPR) promoter. The list of TFs was subjected to enrichment analysis. To evaluate the effects of shortlisted TFs on leptin-LEPR-DHH to induce EMT in LSCs, qPCR and western blotting were performed. For statistical analysis GraphPad Prism was used. For statistical analysis GraphPad Prism was used. All data were presented as the means ± SEM. All data were presented as the means ± SEM Results Over 60 TF’s were identified by UCSC Genome browser, which were subjected to enrichment analysis using ENRICHR and Ingenuity pathway analysis. Output revealed TFs; SOX2, SOX8 and SOX9 that were enriched for mechanisms influencing hormonal regulation via hypothalamic pituitary gonadal axis. The role of these TFs has been well established in epithelial to mesenchymal transition in different cancer types, however their impact on LSC differentiation remained underexplored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8 Sox9 respectively. This was confirmed at RNA as well as at protein levels. Consequently, upon inhibition of LEPR, the expression of SOX2, SOX8 and SOX9 was reduced as well. Furthermore we used SOX8, SOX9 and SOX2 antagonists in the presence or absence of Leptin to validate the effects on EMT of LCs. Conclusions Our results demonstrate that Leptin-LEPR-DHH induced Leydig stem cells differentiation in testis is modulated by EMT inducing transcription factors which binds to LEPR. Further studies are ongoing to explore the potential therapeutic effects of leptin treatment in improving fertility. Disclosure No

METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Pirfenidone suppressed triple-negative breast cancer metastasis by inhibiting the activity of the TGF-β/SMAD pathway.

Among breast cancer patients, metastases are the leading cause of death. Despite decades of effort, little progress has been made to improve the treatment of breast cancer metastases, especially triple-negative breast cancer (TNBC). The extracellular matrix plays an important role in tumour growth and metastasis by causing its deposition, remodelling, and signalling. As we know, the process of fibrosis results in excessive amounts of extracellular matrix being deposited within the cells. So, it will be interesting to study if the use of anti-fibrotic drugs in combination with conventional chemotherapy drugs can produce synergistic antitumor effects. In this study, we assessed the efficacy of Pirfenidone (PFD), an FDA-approved medication for the treatment of idiopathic pulmonary fibrosis, on TNBC cells as well as its anti-tumour effects in xenograft tumour model. PFD inhibited in a dose-dependent manner breast cancer cell proliferation, migration, and invasion, while promoted their apoptosis in vitro. PFD also suppressed TGF-β-induced activation of Smad signalling pathway and expression level of EMT-inducing transcription factors (e.g. SNAI2, TWIST1, ZEB1) as well as the mesenchymal genes such as VIMENTIN and N-Cadherin. On the contrary, the expression level of epithelial marker gene E-Cadherin was up-regulated in the presence of PFD. In vivo, PFD alone exerted a milder but significant anti-tumour effect than the chemotherapy drug nanoparticle albumin-bound paclitaxel (nab-PTX) did in the breast cancer xenograft mouse model. Interestingly, PFD synergistically boosted the cancer-killing effect of nab-PTX. Furthermore, Our data suggest that PFD suppressed breast cancer metastasis by inhibiting the activity of the TGFβ/SMAD pathway.

SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration.

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many carcinomas depend on EMT activation, partial EMT, or hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGFβ signal and EMT-inducing transcription factors, such as ZEB1/2, in tumor cells. However, reverse EMT factors are less studied. We demonstrate that prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1γ. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and β-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway.

MSC-Derived exosomes suppress colorectal cancer cell proliferation and metastasis via miR-100/mTOR/miR-143 pathway

Exosomes derived from mesenchymal stem cells (MSCs) are mostly responsible for the therapeutic effects of MSCs. To show the therapeutic effects of the human bone marrow MSC-derived exosomes (MSC-Exos) on colorectal cancer (CRC) and explore the molecular cross-talks between them, CRC cells were treated with the MSC-Exos. We found that MSC-Exos were enriched with miR-100 and miR-143, which effectively downregulated mTOR, Cyclin D1, K-RAS, HK2 while upregulated p-27 expression. All these effects were reversed by concurrent treatment with MSC-Exos and antagomiR-100, confirming that they were caused by exosomal transfer of miR-100 into recipient CRC cells. Moreover, exosomal miR-100 promoted endogenous miR-143 expression. The flow cytometry, MTT and trypan blue assays revealed that MSC-Exos could efficiently suppress proliferation and induce apoptosis of the CRC cells. Furthermore, wound healing, transwell migration and invasion assays confirmed their inhibitory effects on the migration and invasiveness of SW480 cells. We further confirmed these effects by analyzing the expression levels of epithelial to mesenchymal transition (EMT) factors and metastasis-related genes. Results showed that MSC-Exos significantly suppressed the expression of MMP2 and MMP9 (metastasis-related genes), SNAIL and TWIST (EMT-inducing transcription factors), Vimentin and N-cadherin (mesenchymal cell markers), whereas E-cadherin (epithelial cell marker) was remarkably up-regulated. Collectively, our data indicated that MSC-Exos could suppress proliferation, migration, invasion and metastasis while inducing the apoptosis of the CRC cells via miR-100/mTOR/miR-143 axis. Our findings highlight that MSC-Exo treatment as well as miR-100 restoration might be considered as potential therapeutic strategies for CRC.

Silencing of RNA binding protein, ZFP36L1, promotes epithelial-mesenchymal transition in liver cancer cells by regulating transcription factor ZEB2

RNA binding proteins (RBPs) of the zinc finger protein 36 family including zinc finger protein 36 like 1 (ZFP36L1) are implicated in cancer, however, the underlying molecular mechanisms have remained unclear. These proteins function by regulating post-transcriptional gene expression upon binding to the AU-rich elements (ARE's) within the 3’untranslated regions (3’UTRs) of specific mRNAs and increasing their mRNA turnover. Here, we tested the role of ZFP36L1 in hepatocellular carcinoma (HCC) cell lines. ZFP36L1 was under-expressed among the three RBPs in a majority of the HCC cell lines. Silencing of ZFP36L1 in two of the seven HCC cell lines resulted in epithelial-mesenchymal transition (EMT) like morphological changes, which were characterized by the transition of epithelial morphology to elongated mesenchymal morphology and increased migration and invasion potential. Conversely, overexpression of ZFP36L1 abolished these changes. RNA-seq analysis of ZFP36L1-depleted HCC cells revealed a significant upregulation of an EMT-inducing transcription factor, ZEB2 (zinc-finger E-box-binding homeobox 2), and enrichment of pathways associated with mesenchymal cell development and differentiation. ZEB2 mRNA contains AREs within its 3’UTR and its stability was increased following ZFP36L1 knockdown. Conversely, ZEB2 was significantly downregulated following ZFP36L1 overexpression and ZEB2 3’UTR was regulated by ZFP36L1 in luciferase reporter assays. These data identify ZEB2 mRNA as a ZFP36L1 target in HCC cells and demonstrate that ZFP36L1 regulates EMT possibly through direct regulation of ZEB2 mRNA. In summary, our results demonstrate that ZFP36L1 suppresses EMT inliver cancer cells by down-regulating the expression of EMT-inducing transcription factor, ZEB2. These data suggest an important role of ZFP36L1 in the development, progression, and metastasis of hepatocellular cancer.

Epithelial-Mesenchymal Transition Suppresses AMPK and Sensitizes Cancer Cells to Pyroptosis under Energy Stress.

Epithelial–mesenchymal transition (EMT) is implicated in tumor metastasis and therapeutic resistance. It remains a challenge to target cancer cells that have undergone EMT. The Snail family of key EMT-inducing transcription factors directly binds to and transcriptionally represses not only epithelial genes but also a myriad of additional genomic targets that may carry out significant biological functions. Therefore, we reasoned that EMT inherently causes various concomitant phenotypes, some of which may create targetable vulnerabilities for cancer treatment. In the present study, we found that Snail transcription factors bind to the promoters of multiple genes encoding subunits of the AMP-activated protein kinase (AMPK) complex, and expression of AMPK genes was markedly downregulated by EMT. Accordingly, high AMPK expression in tumors correlated with epithelial cell markers and low AMPK expression in tumors was strongly associated with adverse prognosis. AMPK is the principal sensor of cellular energy status. In response to energy stress, AMPK is activated and critically reprograms cellular metabolism to restore energy homeostasis and maintain cell survival. We showed that activation of AMPK by energy stress was severely impaired by EMT. Consequently, EMT cancer cells became hypersensitive to a variety of energy stress conditions and primarily underwent pyroptosis, a regulated form of necrotic cell death. Collectively, the study suggests that EMT impedes the activation of AMPK signaling induced by energy stress and sensitizes cancer cells to pyroptotic cell death under energy stress conditions. Therefore, while EMT promotes malignant progression, it concurrently induces collateral vulnerabilities that may be therapeutically exploited.

Abstract 1561: Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation

Abstract Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides lacking protein-coding potential, which are involved in diverse pathophysiologic conditions including cancer progression and metastasis. Metastasis is initiated by a cellular process, epithelial-to-mesenchymal transition (EMT), and many lncRNAs can be affected by and participate in the EMT process. To find out lncRNAs regulated by the EMT signals, we performed an NGS-based RNA sequencing in murine lung cancer cells, and finally selected lnc-NR2F1 that is upregulated by ZEB1, an EMT-inducing transcription factor. lnc-NR2F1 expression levels showed a positive correlation with ZEB1 mRNA levels and EMT scores in 13 mouse lung cancer cells or in lung adenocarcinoma data from TCGA. The migration and invasion of lung cancer cells were decreased after lnc-NR2F1 knockdown and increased after lnc-NR2F1 overexpression. lnc-NR2F1 was transcriptionally up-regulated by NR2F1, a transcription factor transcribed from the antisense strand. Interestingly, NR2F1 was transcriptionally induced by ZEB1, and promoted the migration and invasion of lung cancer cells. Through the analysis of public ChIRP-seq data, we found that lnc-NR2F1 can modulate the expression of TWIST2, and add-back of TWIST2 restored the migration and invasion of lung cancer cells suppressed by lnc-NR2F1 knockdown. In conclusion, lnc-NR2F1 is induced by a ZEB1-NR2F1 axis and stimulates the migration and invasion of lung cancer cells via transcriptional regulation of TWIST2. Citation Format: Eun Ju Kim, Young-Ho Ahn. Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1561.

Cordycepin Inhibits Triple-Negative Breast Cancer Cell Migration and Invasion by Regulating EMT-TFs SLUG, TWIST1, SNAIL1, and ZEB1.

Cancer metastasis is the most important cause of cancer-related death, and epithelial-to-mesenchymal transition (EMT) plays crucial roles in cancer metastasis. Cordycepin (CD) is highly enriched in the medicinally used Cordyceps mushroom. In this study, we conducted the antimetastatic activities of CD, specifically focusing on its regulatory effects on EMT-inducing transcription factors (EMT-TFs) in triple-negative breast cancer (TNBC). Our study showed CD to inhibit the growth, migration, and invasion of BT549 and 4T1 cancer cell lines, by employing cell viability assay and real-time cell analyses. The protein levels of N-Cadherin and E-Cadherin, as well as their transcription factors TWIST1, SLUG, SNAIL1, and ZEB1 in BT549 and 4T1 cells, were estimated by Western blot assays. Results from dual-luciferase reporter assays demonstrated that CD is capable of inactivating the EMT signaling pathway by inhibiting TWIST1 and SLUG expression. Furthermore, in vivo studies with mice carrying cancer cell-derived allograft tumors showed the inhibitory effect of CD on cancer cell growth and metastasis. Furthermore, the additive/synergistic anti-metastasis effect of CD and thymoquinone (TQ), another natural product with promising anticancer roles, was demonstrated by combinational treatment. The results from this research indicate that CD would be a promising therapeutic molecule against TNBC by targeting EMT-TFs, possibly in SLUG, TWIST1, SNAIL1, and ZEB1.

SLUG is a key regulator of epithelial-mesenchymal transition in pleomorphic adenoma

AbstractThe histogenesis of pleomorphic adenoma (PA) of the salivary glands remains controversial. PAs are characterized by the transition of epithelial cells to spindled mesenchymal cells, known as epithelial-mesenchymal transition (EMT). The present study aimed to identify a major EMT-inducing transcription factor (EMT-TF) in PAs. Real-time PCR analysis of SNAIL, SLUG, ZEB1, and TWIST1 demonstrated that only SLUG was significantly upregulated in normal salivary glands and PAs. Combined in situ hybridization for SLUG and multiplex immunohistochemistry for CK19 and P63 revealed that SLUG was specifically expressed in the myoepithelial cells of normal salivary glands. In PAs, SLUG was expressed in neoplastic myoepithelial cells and stromal cells but not in the luminal cells lining the inner layers of tumor glands. SLUG expression showed no correlation with PLAG1 expression, and in vitro experiments demonstrated that PLAG1 suppression in primary cultured PA cells or PLAG1 overexpression in HEK 293 T cells did not affect SLUG levels, indicating that PLAG1 was not involved in the upregulation of SLUG in PAs. The suppression of SLUG expression in cultured PA cells resulted in a morphology change to a less elongated shape and attenuated tumor growth. In addition, SLUG downregulation led to increased E-cadherin and decreased N-cadherin and vimentin expression levels along with decreased migratory activity in cultured PA cells. These findings suggest that SLUG is a major TF that can induce EMT in PAs. In summary, SLUG is specifically and highly expressed in the myoepithelial cells and stromal cells of PAs and is a key regulator of EMT in PAs.

Induction of mesenchymal-epithelial transition (MET) by epigallocatechin-3-gallate to reverse epithelial-mesenchymal transition (EMT) in SNAI1-overexpressed renal cells: A potential anti-fibrotic strategy

Dynamic transdifferentiation of epithelial cells from epithelial-mesenchymal transition (EMT) to its reverse process, mesenchymal-epithelial transition (MET), has gained wide attention for management of cancers and tissue fibrosis. In this study, we addressed beneficial effects of epigallocatechin-3-gallate (EGCG) on EMT-MET reversion using an in vitro EMT model by overexpressing SNAI1 gene encoding Snail1, an EMT-inducing transcription factor, into renal tubular epithelial cells (pcDNA6.2-SNAI1 cells). The cells transfected with empty vector (pcDNA6.2 cells) served as the control. Titrating EGCG concentrations revealed its optimal dose at 25 µM for 24-h, which was used throughout. pcDNA6.2-SNAI1 cells had increased spindle index and typical morphology of EMT, whereas EGCG could restore the normal index and morphology. Increased nuclear Snail1 and β-catenin; increased cytoplasmic Snail1, p-GSK-3β, vimentin, fibronectin and F-actin; and decreased occludin, ZO-1, transepithelial resistance (TER), E-cadherin and cell cluster size were observed in the pcDNA6.2-SNAI1 cells. These pcDNA6.2-SNAI1 cells also had increased migrating activity associated with increased forward but decreased non-forward α-tubulin filaments, G0/G1 cell cycle escape, and increased matrix metalloproteinase-2 (MMP-2) and MMP-9. All of these EMT features were successfully abolished by EGCG (partially, completely, or overly). Collectively, our data have demonstrated that EGCG can reverse EMT to MET processes in renal cells. Therefore, EGCG may have the therapeutic potential as one of the promising anti-fibrotic agents to reverse the fibrotic kidney.

Leptin-LEPR-Desert Hedgehog Signaling Induced Leydig Stem Cells Differentiation in Testis is Modulated by EMT Inducing Transcription Factors which Binds to LEPR

ABSTRACT Introduction Leydig cells (LCs) are the primary source of testosterone (T) in men, and their dysfunction can lead to T deficiency. The growth and differentiation of LCs is influenced by the paracrine factors released by the testicular microenvironment (TME). Leptin is a 16-kDa protein produced primarily by adipose tissue and exerts a significant influence on hormonal regulation. In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cell (LSCs) differentiation and subsequent T production under the regulation of the desert hedgehog signaling pathway. Objective To gain an in-depth understanding of mechanisms that derive the Leptin secretion and decide the fate of LCs are still unclear and is the focus of the present study. Methods A total of 13 men with testicular failure underwent testis biopsies. The testicular cells were characterized using Flow cytometry, Immunostaining and RNA sequencing then the cells were also treated with Leptin in a dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) manner. For the functional studies, UCSC genome browser was used to study the transcription factor genes (TFs) binding the leptin receptor (LEPR) promoter. The list of TFs was subjected to enrichment analysis. To evaluate the effects of shortlisted TFs on leptin-LEPR-DHH to induce EMT in LSCs, qPCR and western blotting were performed. For statistical analysis GraphPad Prism was used. For statistical analysis GraphPad Prism was used. All data were presented as the means ± SEM. All data were presented as the means ± SEM. Results UCSC Genome browser revealed over 50 TFs, which were subjected to enrichment analysis using ENRICHR. Output revealed TFs; SOX2, SOX8 and SOX9 that were enriched for mechanisms influencing hormonal regulation via hypothalamic pituitary gonadal axis. The role of these TFs has been well established in epithelial to mesenchymal transition in different cancer types, however their impact on LSC differentiation remained underexplored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8, Sox9 respectively. This was confirmed at RNA as well as at protein levels. Consequently, upon inhibition of LEPR, the expression of SOX2, SOX8 and SOX9 was reduced as well. Moreover, leptin treatment in conjunction with SOX2, SOX8 and SOX9 was found to modulate epithelial to mesenchymal transition in LCs. Conclusions Our results demonstrate that Leptin-LEPR-DHH induced Leydig stem cells differentiation in testis is modulated by EMT inducing transcription factors which binds to LEPR. Further studies are ongoing to explore the potential therapeutic effects of leptin treatment in improving fertility. Financial Support: Supported by the Clinician Scientist Development award from American Cancer Society to RR and Research Scholar Award from American Urological Association to HA. J.M.H. is supported by NIH grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, 5UM1 HL113460 and Soffer Family Foundation Disclosure No

The Fra-1/AP-1 Oncoprotein: From the “Undruggable” Transcription Factor to Therapeutic Targeting

Simple SummaryCancer-associated mortality largely depends on metastatic dissemination. Metastasizing cancer cells exhibit drastic phenotypic changes, including the ability to migrate, invade surrounding tissues, survive in the bloodstream, adapt to different microenvironments, and resist to therapeutic treatments. These changes depend on the genetic reprogramming orchestrated by relatively few players (transcription factors). Among the components of the dimeric transcription factor AP-1, the nuclear oncoprotein Fra-1 is strongly implicated in metastasis mechanisms. Therefore, Fra-1, along with other proinvasive transcription factors, represents an ideal therapeutic target. However, as for other DNA-binding proteins, the design of inhibitory drugs is hampered by the structural features of Fra-1. In this Review, after summarizing the Fra-1 functions and mechanisms of accumulation in invasive tumors, we survey the possible application of multiple strategies and emerging technologies aimed at the inhibition of Fra-1 expression and activity, to prevent metastatic dissemination and therapeutic resistance. The genetic and epigenetic changes affecting transcription factors, coactivators, and chromatin modifiers are key determinants of the hallmarks of cancer. The acquired dependence on oncogenic transcriptional regulators, representing a major determinant of cancer cell vulnerability, points to transcription factors as ideal therapeutic targets. However, given the unavailability of catalytic activities or binding pockets for small-molecule inhibitors, transcription factors are generally regarded as undruggable proteins. Among components of the AP-1 complex, the FOS-family transcription factor Fra-1, encoded by FOSL1, has emerged as a prominent therapeutic target. Fra-1 is overexpressed in most solid tumors, in response to the BRAF-MAPK, Wnt-beta-catenin, Hippo-YAP, IL-6-Stat3, and other major oncogenic pathways. In vitro functional analyses, validated in onco-mouse models and corroborated by prognostic correlations, show that Fra-1-containing dimers control tumor growth and disease progression. Fra-1 participates in key mechanisms of cancer cell invasion, Epithelial-to-Mesenchymal Transition, and metastatic spreading, by driving the expression of EMT-inducing transcription factors, cytokines, and microRNAs. Here we survey various strategies aimed at inhibiting tumor growth, metastatic dissemination, and drug resistance by interfering with Fra-1 expression, stability, and transcriptional activity. We summarize several tools aimed at the design and tumor-specific delivery of Fra-1/AP-1-specific drugs. Along with RNA-based therapeutics targeting the FOSL1 gene, its mRNA, or cognate regulatory circRNAs, we will examine the exploitation of blocking peptides, small molecule inhibitors, and innovative Fra-1 protein degraders. We also consider the possible caveats concerning Fra-1 inhibition in specific therapeutic contexts. Finally, we discuss a recent suicide gene therapy-based approach, aimed at selectively killing the Fra-1-overexpressing neoplastic cells.