Expression Of EMT Markers Research Articles

Growth differentiation factor 15 is a glucose-downregulated gene acting as the crosstalk between stroma and cancer cells of the human bladder.

Hyperglycemia and hyperglycosuria, two primary characteristics of diabetes mellitus, may increase the risk of cancer initiation, particularly for bladder cancer. The effectiveness of metformin, a common antidiabetic agent, is determined by its ability to induce GDF15. However, the mechanism of the GDF15 in relation to glucose, which influences the tumor microenvironment in the human bladder, is not fully understood. This study explores the potential roles of GDF15 in response to glucose in the human bladder. High glucose treatment (30 mM) enhanced phosphorylation of AKT at S473 and AMPK1/2 at S485 to block the counteracting effect of metformin on the AMPK activity in bladder cancer and stroma (HBdSF and HBdSMC) cells compared to normal glucose treatment (5 mM). Metformin modulated the expressions of GDF15, NDRG1, Maspin, and EMT markers to attenuate cell proliferation and invasion of bladder cancer cells. CAPE, like metformin, behaves as an inducer of AMPK activity to stimulate GDF15 expression. Knockdown of GDF15 blocked the downregulation of CAPE on the contraction of HBdSMC cells. Both CAPE-induced GDF15 expression and the supernatant from bladder cancer cells with overexpressing GDF15 impeded the HBdSF and HBdSMC cell migration, suggesting that CAPE-upregulated GDF15 blocked the cell migration. These findings reveal that high glucose treatment inhibits the counteracting effects of either metformin or CAPE on the AMPK activity, and GDF15 is downregulated by glucose and induced by metformin and CAPE in both stroma and cancer cells. Furthermore, GDF15 is an antitumor gene facilitating communication between stroma and cancer cells in the human bladder.

Melittin Inhibits Colorectal Cancer Growth and Metastasis by Ac-Tivating the Mitochondrial Apoptotic Pathway and Suppressing Epithelial-Mesenchymal Transition and Angiogenesis.

Melittin has previously been found to have a positive effect on colorectal cancer (CRC) treatment, one of the most difficult-to-treat malignancies, but the mechanism by which this effect occurs remains unclear. We evaluated melittin's pro-apoptotic and anti-metastatic effects on CRC in vitro and in vivo. The results showed that melittin-induced mitochondrial ROS bursts decreased ΔΨm, inhibited Bcl-2 expression, and increased Bax expression in both cells and tumor tissues. This led to increased mitochondrial membrane permeability and the release of pro-apoptotic factors, particularly the high expression of Cytochrome C, initiating the apoptosis program. Additionally, through wound-healing and transwell assays, melittin inhibited the migration and invasion of CRC cells. In vivo, the anti-metastatic effect of melittin was also verified in a lung metastasis mouse model. Western blotting and immunohistochemistry analysis indicated that melittin suppressed the expression of MMPs and regulated the expression of crucial EMT markers and related transcription factors, thereby inhibiting EMT. Furthermore, the melittin disrupts neovascularization, ultimately inhibiting the metastasis of CRC. In conclusion, melittin exerts anti-CRC effects by promoting apoptosis and inhibiting metastasis, providing a theoretical basis for further research on melittin as a targeted therapeutic agent for CRC.

Prenatal exposure to di-n-butyl phthalate promotes RIPK1-regulated necroptosis of uroepithelial cells and induces hypospadias through the epithelial-mesenchymal transition process in newborn male rats

Maternal exposure to di-n-butyl phthalate (DBP) has been linked to the induction of hypospadias; however, the underlying mechanism remains unclear. Necroptosis is reported to be implicated in developmental malformations. This study aimed to investigate the underlying mechanism of necroptosis in the development of hypospadias. DBP was dissolved in corn oil, and pregnant rats were administered a precisely measured dose of DBP (750 mg/kg/day) via gastric intubation from gestation day 14–18. Control rats received only corn oil. The day of birth was considered postnatal day (PND) 1. Male hypospadias rats were identified on PND 7. Genital tubercle tissues were collected and stored at −80°C for subsequent PCR analysis, cryopreserved in liquid nitrogen for western blot, or fixed in formalin for immunohistochemistry (IHC) staining. IHC staining and western blot analysis revealed increased expression of RIPK1 and necroptosis markers in genital tubercle (GT) tissue compared to the control group. Additionally, higher levels of EMT and impaired androgen receptor expression were observed in GT tissue. Exposure to increased DBP concentrations in rat primary uroepithelial cells (PUCs) led to elevated ROS production. Necroptosis markers and EMT expression levels were upregulated in PUCs following DBP incubation. Notably, treatment with DBP combined with necrostatin-1, a necroptosis inhibitor, reduced the expression of EMT markers and ROS production compared to DBP treatment alone. In vitro studies further revealed that DBP-induced necroptosis promoted the degradation of E-cadherin through the ubiquitin-proteasome pathway in PUCs. Our findings suggest that maternal exposure to DBP promotes necroptosis in uroepithelial cells by elevating ROS level and EMT status. Thus, necroptosis may play an essential role in the development of hypospadias.

Niraparib Enhances and Synergizes with Ganglioside GD2 to Potentiate its Inhibitory Effect on Bladder Cancer Cells.

This study aimed to study the inhibitory effect of niraparib alone or in combination with GD2 specific antibody on Bladder Cancer (BCa). The migration ability of BCa cells was assessed through a scratch assay. CCK-8 assay was performed to evaluate the viability of BCa cells, and Transwell invasion assays were utilized to examine invasive capacity. The expression levels of E-cadherin and vimentin in BCa cells were measured using QRT-PCR. Western blot showed the EMT level to be the lowest in the niraparib+GD2 group. The transwell invasion assay suggested that the invasion ability of BCa cells was weakened in the niraparib+ GD2 group. CCK8 assay indicated that the proliferation ability of BCa cells was decreased. Scratch test suggested that the migration ability of BCa cells was weakened. PCR result showed that the niraparib + GD2 group had the most significant inhibitory effect on mRNA expression of EMT markers. Niraparib combined with a GD2-specific antibody exerted a more prominent inhibitory effect on BCa.

Differential effects of hypoxia on motility using various in vitro models of lung adenocarcinoma

Lung cancer is the leading cause of cancer-related death globally. Metastasis is the most common reason of mortality in which hypoxia is suggested to have a pivotal role. However, the effect of hypoxia on the metastatic potential and migratory activity of cancer cells is largely unexplored and warrants detailed scientific investigations. Accordingly, we analyzed changes on cell proliferation and migratory activity both in single-cell migration and invasion under normoxic and hypoxic conditions in lung adenocarcinoma cell lines. Alterations in crucial genes and proteins associated with cellular response to hypoxia, epithelial-mesenchymal transition, proliferation and apoptosis were also analyzed. Generally, we observed no change in proliferation upon hypoxic conditions and no detectable induction of apoptosis. Interestingly, we observed that single-cell motility was generally reduced while invasion under confluent conditions using scratch assay was enhanced by hypoxia in most of the cell lines. Furthermore, we detected changes in the expression of EMT markers that are consistent with enhanced motility and metastasis-promoting effect of hypoxia. In summary, our study indicated cell line-, time of exposure- and migrational type-dependent effects of hypoxia in cellular proliferation, motility and gene expression. Our results contribute to better understanding and tackling cancer metastasis.

METTL3-Regulated lncRNA SNHG7 Drives MNNG-Induced Epithelial-Mesenchymal Transition in Gastric Precancerous Lesions.

As a representative item of chemical carcinogen, MNNG is closely associated with the onset of gastric cancer (GC), where N6-methyladonosine (m6A) RNA methylation is recognized as a critical epigenetic event. In our previous study, we found that the m6A modification by methyltransferase METTL3 was up-regulated in MNNG-exposed malignant GES-1 cells (MC cells) compared to control cells in vitro, and long non-coding RNA SNHG7 as a downstream target of the METTL3. However, the functional role of METTL3 in mediating the SNHG7 axis in MNNG-induced GC remains unclear. In the present study, we continuously investigate the functional role of METTL3 in mediating the SNHG7 axis in MNNG-induced GC. RIP-PCR and m6A-IP-qPCR were used to examine the molecular mechanism underlying the METTL3/m6A/SNHG7 axis in MNNG-induced GC. A METTL3 knockout mice model was constructed and exposed by MNNG. Western blot analysis, IHC analysis, and RT-qPCR were used to measure the expression of METTL3, SNHG7, and EMT markers. In this study, we demonstrated that in MNNG-induced GC tumorigenesis, the m6A modification regulator METTL3 facilitates cellular EMT and biological functions through the m6A/SNHG7 axis using in vitro and in vivo models. In conclusion, our study provides novel insights into critical epigenetic molecular events vital to MNNG-induced gastric carcinogenesis. These findings suggest the potential therapeutic targets of METTL3 for GC treatment.

Wedelolactone suppresses breast cancer growth and metastasis via regulating TGF-β1/Smad signaling pathway.

Breast cancer is a malignant tumor with high invasion and metastasis. TGF-β1-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of breast cancer. Wedelolactone (Wed) is extracted from herbal medicine Ecliptae Herba, which is reported to have antineoplastic activity. Here, we aimed to elucidate the efficacy and mechanism of Wed against breast cancer. The effects of Wed on migration and invasion of 4T1 were detected. The expression of EMT-related markers was detected by Western blot and qPCR. The 4T1 orthotopic murine breast cancer model was established to evaluate the therapeutic effect of Wed on the growth and metastasis of breast cancer through TGF-β1/Smad pathway. Wed inhibited the proliferation, migration and invasion of 4T1. It exhibited concentration-dependent inhibition of p-Smad2/3. Wed also reversed the expression of EMT-markers induced by TGF-β1. In addition, Wed suppressed the growth and metastasis of breast cancer in mice. It also affected p-Smad3 expression as well as EMT-related genes, suggesting that its anti-breast cancer effect may be related to the TGF-β1/Smad pathway. Wed reverses EMT by regulating TGF-β1/Smad pathway, potentially serving as a therapeutic agent for breast cancer. Wed is expected to be a potential drug to inhibit TGF-β1/Smad pathway-related diseases.

MCTP1 increases the malignancy of androgen-deprived prostate cancer cells by inducing neuroendocrine differentiation and EMT.

Neuroendocrine prostate cancer (PCa) (NEPC), an aggressive subtype that is associated with poor prognosis, may arise after androgen deprivation therapy (ADT). We investigated the molecular mechanisms by which ADT induces neuroendocrine differentiation in advanced PCa. We found that transmembrane protein 1 (MCTP1), which has putative Ca2+ sensing function and multiple Ca2+-binding C2 domains, was abundant in samples from patients with advanced PCa. MCTP1 was associated with the expression of the EMT-associated transcription factors ZBTB46, FOXA2, and HIF1A. The increased abundance of MCTP1 promoted PC3 prostate cancer cell migration and neuroendocrine differentiation and was associated with SNAI1-dependent EMT in C4-2 PCa cells after ADT. ZBTB46 interacted with FOXA2 and HIF1A and increased the abundance of MCTP1 in a hypoxia-dependent manner. MCTP1 stimulated Ca2+ signaling and AKT activation to promote EMT and neuroendocrine differentiation by increasing the SNAI1-dependent expression of EMT and neuroendocrine markers, effects that were blocked by knockdown of MCTP1. These data suggest an oncogenic role for MCTP1 in the maintenance of a rare and aggressive prostate cancer subtype through its response to Ca2+ and suggest its potential as a therapeutic target.

α5-nAChR/ADAM10 signaling mediates nicotine-related cutaneous melanoma progression via STAT3 activation.

Skin cutaneous melanoma (SKCM) is the skin malignancy with the highest mortality rate, and its morbidity rate is on the rise worldwide. Smoking is an independent marker of poor prognosis in melanoma. The α5-nicotinic acetylcholine receptor (α5-nAChR), one of the receptors for nicotine, is involved in the proliferation, migration and invasion of SKCM cells. Nicotine has been reported to promote the expression of a disintegrin and metalloproteinase 10 (ADAM10), which is the key gene involved in melanoma progression. Here, we explored the link between α5-nAChR and ADAM10 in nicotine-associated cutaneous melanoma. α5-nAChR expression was correlated with ADAM10 expression and lower survival in SKCM. α5-nAChR mediated nicotine-induced ADAM10 expression via STAT3. The α5-nAChR/ADAM10 signaling axis was involved in the stemness and migration of SKCM cells. Furthermore, α5-nAChR expression was associated with ADAM10 expression, EMT marker expression and stemness marker expression in nicotine-related mice homograft tissues. These results suggest the role of the α5-nAChR/ADAM10 signaling pathway in nicotine-induced melanoma progression.

Abstract PO3-24-08: Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells

Abstract Purpose Cancer stem cells are considered to cause recurrence and metastasis due to their resistance to drug and radiation therapy. Breast cancer stem cells are therefore the true target for the treatment of breast cancer. In this study, we established a method to selectively culture breast cancer stem cells from patient-derived breast cancer tissue, and we analyzed their characteristics using immunostaining to identify gene expression. Method Breast cancer tissues from ER+, HER2- and drug-naive patients were fragmented and cultured using low-adherent plates (spheroid culture). We investigated the stemness of the breast cancer cells obtained by the spheroid culture by immunostaining the breast cancer stem cell markers and examining their proliferation and differentiation in a mouse transplantation model. The diversity of the cells was further examined by means of the expression of the EMT markers and genetic analysis. Results In 48 cases, the proportion of CD44+/CD24- breast cancer cells increased from 13.8% to 61.6% in the spheroid culture. In addition, even small numbers of cells obtained by the spheroid culture were transplanted into mice, and the transplanted cells subsequently differentiated in the mice, demonstrating their stemness. ER expression was negatively changed in 52.1% of cases, and the expression of EMT markers, Twist, Snail, and Vimentin, was increased from 43.8% to 75.0%, 12.9% to 58.1%, and 7.71% to 37.7%, respectively. A DNA microarray with 14 cases showed that breast cancer stem cells obtained from ER+ and HER2- breast cancer tissue were classified into two groups. Conclusion We demonstrated that breast cancer stem cells were selectively cultured from patient-derived breast cancer tissue in a spheroid culture. Furthermore, breast cancer stem cells have different characteristics from breast cancer cells in primary tumors and are diverse at an EMT level, and in genetic analyses. Table. Expression of primary tumors and spheroids Citation Format: Satoshi Sueoka, Takayuki Kadoya, Kai Azusa, Shinsuke Sasada, Akiko Emi, Morihito Okada, Yukino Kobayashi, Koh Nakayama. Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells [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 PO3-24-08.

Melatonin prevents pulmonary fibrosis caused by PM2.5 exposure by targeting epithelial-mesenchymal transition

Pulmonary fibrosis is a lung disorder characterized by the accumulation of abnormal extracellular matrix, scar tissue formation, and tissue stiffness. Type II alveolar epithelial cells (AEII) play a critical role in repairing lung tissue after injury, and repeated injury to these cells is a key factor in the development of pulmonary fibrosis. Chronic exposure to PM2.5, a type of air pollution, has been shown to increase the incidence and severity of pulmonary fibrosis by enhancing the activation of EMT in lung epithelial cells. Melatonin, a hormone with antioxidant properties, has been shown to prevent EMT and reduce fibrosis in previous studies. However, the mechanism through which melatonin targets EMT to prevent pulmonary fibrosis caused by PM2.5 exposure has not been extensively discussed before. In this current study, we found that melatonin effectively prevented pulmonary fibrosis caused by prolonged exposure to PM2.5 by targeting EMT. The study demonstrated changes in cellular morphology and expression of EMT markers. Furthermore, the cell migratory potential induced by prolonged exposure to PM2.5 was greatly reduced by melatonin treatment. Finally, in vivo animal studies showed reduced EMT markers and improved pulmonary function. These findings suggest that melatonin has potential clinical use for the prevention of pulmonary fibrosis.

STAT1 mediated downregulation of the tumor suppressor gene PDCD4, is driven by the atypical cadherin FAT1, in glioblastoma

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. It has been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression.

Cancer-associated fibroblasts drive colorectal cancer cell progression through exosomal miR-20a-5p-mediated targeting of PTEN and stimulating interleukin-6 production.

This study evaluated the clinical relevance of a set of five serum-derived circulating microRNAs (miRNAs) in colorectal cancer (CRC). Additionally, we investigated the role of miR-20a-5p released by exosomes derived from cancer-associated fibroblasts (CAFs) in the context of CRC. The expression levels of five circulating serum-derived miRNAs (miR-20a-5p, miR-122-5p, miR-139-3p, miR-143-5p, and miR-193a-5p) were quantified by real-time quantitative PCR (RT-qPCR), and their associations with clinicopathological characteristics in CRC patients were assessed. The diagnostic accuracy of these miRNAs was determined through Receiver Operating Characteristic (ROC) curve analysis. CAFs and normal fibroblasts (NFs) were isolated from tissue samples, and subsequently, exosomes derived from these cells were isolated and meticulously characterized using electron microscopy and Western blotting. The cellular internalization of fluorescent-labeled exosomes was visualized by confocal microscopy. Gain- and loss-of-function experiments were conducted to elucidate the oncogenic role of miR-20a-5p transferred by exosomes derived from CAFs in CRC progression. The underlying mechanisms were uncovered through luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assays, as well as proliferation and migration assays. The expression levels of serum-derived circulating miR-20a-5p and miR-122-5p were significantly higher in CRC and were positively correlated with advanced stages of tumorigenesis and lymph node metastasis (LNM). In contrast, circulating miR-139-3p, miR-143-5p, and miR-193a-5p were down-regulated in CRC and associated with early tumorigenesis. Except for miR-139-3p, they showed a negative correlation with LNM status. Among the candidate miRNAs, significantly elevated levels of miR-20a-5p were observed in both cellular and exosomal fractions of CAFs. Our findings indicated that miR-20a-5p induces the expression of EMT markers, partly by targeting PTEN. Exosomal miR-20a secreted by CAFs emerged as a key factor enhancing the proliferation and migration of CRC cells. The inhibition of miR-20a impaired the proliferative and migratory potential of CAF-derived exosomes in SW480 CRC cells, suggesting that the oncogenic effects of CAF-derived exosomes are mediated through the exosomal transfer of miR-20a. Furthermore, exosomes originating from CAFs induced increased nuclear translocation of the NF-kB p65 transcription factor in SW480 CRC cells, leading to increased interleukin-6 (IL-6) production. We established a set of five circulating miRNAs as a non-invasive biomarker for CRC diagnosis. Additionally, our findings shed light on the intricate mechanisms underpinning the oncogenic impacts of CAF-derived exosomes and underscore the pivotal role of miR-20a-5p in CRC progression.

Network pharmacology-based investigation of the effects of Shenqi Fuzheng injection on glioma proliferation and migration via the SRC/PI3K/AKT signaling pathway

Ethnopharmacological relevanceIn the clinic, Shenqi Fuzheng Injection (SFI) is used as an adjuvant for cancer chemotherapy. However, the molecular mechanism is unclear. Aim of the studyWe screened potential targets of SFI action on gliomas by network pharmacology and performed experiments to validate possible molecular mechanisms against gliomas. Materials and methodsWe consulted relevant reports on the SFI and glioma incidence from PubMed and Web of Science and focused on the mechanism through which the SFI inhibits glioma. According to the literature, two primary SFI components—Codonopsis pilosula (Franch.) Nannf. and Astragalus membranaceus (Fisch.) Bunge—have been found. All plant names have been sourced from "The Plant List" (www.theplantlist.org). The cell lines U87, T98G and GL261 were used in this study. The inhibitory effects of SFI on glioma cells U87 and T98G were detected by CCK-8 assay, EdU, plate cloning assay, scratch assay, Transwell assay, immunofluorescence, flow cytometry and Western blot. A subcutaneous tumor model of C57BL/6 mice was constructed using GL261 cells, and the SFI was evaluated by HE staining and immunohistochemistry. The targets of glioma and the SFI were screened using network pharmacology. ResultsA total of 110 targets were enriched, and a total of 26 major active components in the SFI were investigated. There were a total of 3,343 targets for gliomas, of which 79 targets were shared between the SFI and glioma tissues. SFI successfully prevented proliferation and caused cellular S-phase blockage in U87 and T98G cells, thus decreasing their growth. Furthermore, SFI suppressed cell migration by downregulating EMT marker expression. According to the results of the in vivo tests, the SFI dramatically decreased the development of tumors in a transplanted tumour model. Network pharmacological studies revealed that the SRC/PI3K/AKT signaling pathway may be the pathway through which SFI exerts its anti-glioma effects. ConclusionsThe findings revealed that the SRC/PI3K/AKT signaling pathway may be involved in the mechanism through which SFI inhibits the proliferation and migration of glioma cells.

Abstract 6809: Tumor-associated macrophages promote bladder cancer cell migration and invasion through activation of CCL20-CCR6 axis

Abstract We focused on Tumor-Associated Macrophages (TAM) and explored its effects on bladder cancer (BC). Co-culturing PMA-treated THP-1 with BC cell lines (UMUC3 and T24) induced THP-1 to develop suppressed CD68 and increased CD206 expression which differentiated into M2-like macrophages. Co-culture of BC with TAM of this M2-like macrophage enhanced the migration and invasive activities of BC, as well as the expression of EMT marker. Human cytokine antibody array of conditioned medium from the co-culture of BC and TAM showed high CCL20, CCL2 and CXCL7. qPCR revealed that these chemokines were mainly derived from TAMs, not BC. Only CCL20 enhanced the migration and invasion of BC and the expression of EMT markers when each of these three recombinant chemokines was administered to BC. Furthermore, inhibiting CCR6, a receptor specific for CCL20, suppressed migration and invasion as expected. In BC, TAMs promote progression and metastasis by secreting CCL20, and inhibition of CCR6 may be one potential therapeutic approach to suppress BC activity. Citation Format: Ryunosuke Nakagawa, Izumi Kouji, Ren Toriumi, Shuhei Aoyama, Taiki Kamjima, Hiroshi Kano, Tomoyuki Makino, Renato Naito, Hiroshi Yaegashi, Takahiro Nohara, Hiroki Nakata, Atsushi Mizokami. Tumor-associated macrophages promote bladder cancer cell migration and invasion through activation of CCL20-CCR6 axis [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 6809.

Abstract 6814: E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma

Abstract Melanoma is a cancer that is metastatic, drug-refractory and fatal if managed late or inadequately. The transcription factor E2F1 is a key player in melanoma pathology, promoting metastatic traits like EMT, neoangiogenesis, chemoresistance and evasion of immune surveillance. Particularly, high levels of E2F1 trigger tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a prometastatic gene regulatory network (GRN). We aimed to investigate the extent to which E2F1-expressing melanoma cells influence the immune response. Methods: We combined transcriptomic approaches with bioinformatics and structural modeling to search for target genes involved in E2F1-activated immunomodulatory and prometastatic GRNs in aggressive melanoma. Immunoprecipitation and ChIP were performed to verify both PPI and protein-DNA interactions. Functional analyses, including qRT-PCR, immunoblotting and luciferase assays, were performed to verify expression and regulation of downstream targets. Additionally, we established a melanoma-immune cell coculture system with patient-derived CD4+ and CD8+ T cells. Cytokine arrays and ELISA were used for measurement of cytokine secretion to analyze the E2F1-induced tumor-immuno-crosstalk. Results: We identified an E2F1-dependent GRN with IL-6 as a central factor mediating EMT and immune regulation. Mechanistically, E2F1-induced activation and secretion of IL-6 leads to an autocrine inflammatory forward-feedback loop, which in turn further promotes invasiveness and EMT marker expression in melanoma cells. Interestingly, IL6 activated STAT3 associates with E2F1 in a structure-dependent manner, forming a complex that enhances IL-6 expression by binding to a composite E2F1:STAT3-responsive promoter element. Moreover, we observed that the E2F1-IL6 axis dispenses immunomodulatory effects on surrounding CD4+ and CD8+ T cells, further stimulating IL-6 release, but also leading to secretion of IL-10 into the TME. Although IL-10 has an immunosuppressive function, it also showed anti-metastatic properties in melanoma cells. Thus, in contrast to IL-6, IL-10 decreased cell motility and reduced the expression of Vim, Slug, and Snai1, while simultaneous knockdown of E2F1 increased these effects. The findings are supported by clinical data sets showing that melanoma patients who have high levels of E2F1 and IL-6 and low IL-10 exhibit poor survival. Conclusion: Our results unveil a novel gene regulatory program by which E2F1-associated IL-6 release induces the formation of an E2F1:STAT3 complex, resulting in a forward-feedback loop that promotes cancer invasion and regulates T cells response through intercellular cytokine signaling in the tumor microenvironment. Citation Format: Prabir Dhar, Krishna P. Singh, Shailendra K. Gupta, Dhanya Peringot, Alf Spitschak, Brigitte M. Pützer. E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma [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 6814.

Abstract 1791: Metabolic rewiring promotes metastatic potential upon glutamine deprivation in STK11 null KRAS-driven lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) with concurrent oncogenic KRAS and STK11 loss-of-function mutations define an aggressive subtype characterized, in part, by increased metastasis. Loss of STK11, a tumor suppressor and nutrient sensor, leads to dysregulation of many critical cellular processes, including metabolism. As such, STK11 null cancers are “glutamine addicted” to support proliferative properties and the tumor microenvironment therefore becomes glutamine depleted. We hypothesize that conditions of glutamine stress promote metastatic potential in STK11 null KRAS-driven LUAD due to metabolic rewiring. To test this hypothesis, we utilized a cell culture model of KRAS-driven LUAD cell lines and corresponding STK11 knockout cell lines (∆STK11). Parental and ∆STK11 cells underwent the Seahorse mitochondrial stress test in full and glutamine-deficient media to obtain parameters of mitochondrial respiration at baseline and under conditions reflective of the tumor microenvironment, respectively. At baseline, ∆STK11 cells had a hypermetabolic phenotype associated with enhanced glutamine utilization. Deprivation of glutamine resulted in decreased mitochondrial respiration along with a significant increase in live, detached ∆STK11 cells compared to the parental line. Upon further examination, the detached ∆STK11 cells maintained the ability to re-adhere and proliferate in full media while the detached parental cells did not. Additionally, expression of pro-survival, anti-apoptotic and EMT markers in ∆STK11 cells were increased upon glutamine deprivation. To determine the mechanism(s) underlying this pro-metastatic phenotype, we employed heavy nitrogen labeling which revealed an upregulation of the hexosamine biosynthetic pathway (HBP). The HBP is an offshoot of glycolysis that serves as a central hub to regulate many cancer fitness pathways via O-GlcNAcylation; the addition of a GlcNAc moiety to serine/threonine residues of target proteins. Further assessment of O-GlcNAcylation levels via western and far western blot analysis revealed that parental cells had decreasing HBP flux concordant with decreasing glutamine concentration while ∆STK11 cells, conversely, had enhanced HBP flux upon decreasing glutamine availability. These observations suggest ∆STK11 cells utilize the HBP as a protective shunt under decreased glutamine availability. We are currently characterizing the invasive potential and anchorage-independence of parental and ∆STK11 LUAD cells upon glutamine deprivation. Future studies aim to establish the impact of HBP perturbation, both genetically and pharmacologically, on the described measures of metastatic potential. Overall, this work reveals novel insight into the molecular mechanisms altered downstream of STK11 loss that link glutamine metabolism with metastatic properties in KRAS-driven LUAD. Citation Format: Shannon Prior, Logan Sands, Sean Lenahan, Hailey Sarausky, Melissa Scheiber, David Seward, Paula Deming. Metabolic rewiring promotes metastatic potential upon glutamine deprivation in STK11 null KRAS-driven 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 1791.

Abstract 4436: Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics

Abstract Introduction: The high chemoresistance of pancreatic ductal adenocarcinoma (PDAC) is partly attributed to the phenotypic plasticity and high adaptability of PDAC cells to the challenges of the harsh microenviroment. Phenotypic presentation of the PDAC cell is also determined by the lipid composition of the plasma membrane and lipid species that organize the rigid or more flexible lipid bilayer and determine the cellular mobility and plasticity. Lipids are also important energy and signaling molecules that contribute to the aggressiveness of the cell. Here we focus on differences in lipid profile of PDAC cells in epithelial and mesenchymal cell state using single-cell lipidomics approach (SpaceM). Methods: Lipid profile of 2D cultures of eight conventional and ten primary patient derived cell lines of different source (patient derived xenograft, ascites, primary tumor, organoid derived) was investigated. Epithelial (E) or mesenchymal (M) cell state was evaluated by gene and protein expression of EMT markers. Matrix-Assisted Laser Desorption/Ionization (MALDI)-imaging MS was used for data acquisition on positive ion mode at 25 µm step size and m/z range of 600-1000. For annotating lipids in MALDI-imaging data, we used the METASPACE cloud software. For constructing single-cell lipidomic profiles we used the SpaceM method. In total, around 150.000 cells were analyzed and 1400 lipid ions detected. Results: Gene expression analysis suggested prominent metabolism of structural lipids in the conventional epithelial cells. In UMAP analysis of single cell lipidomics data, each cell line, conventional and primary, presented a unique lipid fingerprint with however prominent clustering into the two phenotypes, epithelial and mesenchymal. We detected enrichment of alkyl-acyl phosphatidylcholines, alkyl-acyl phospatidylethanolamines, ceramides and sphingolipids in the epithelial phenotype. Mesenchymal phenotype was enriched for lipids involved in lateral diffusion and low bilayer thickness supporting the increased fluidity of the mesenchymal cell membrane. Furthermore, mesenchymal phenotype was enriched for lipid droplets and energy storing triacylglycerols, also confirmed by histological lipid droplet staining. Conclusions: Using the single cell lipid information, a successful grouping to epithelial or mesenchymal subtype could be performed regardless of sample source, suggesting a conserved lipid profile typical for the two states. Further detailed investigation of single cell lipidomics data aims to unveil the prominent epithelial-mesenchymal state lipid markers and open a niche for functional investigations of cell-state specific markers and potential cell-state targeting. Citation Format: Marija Trajkovic-Arsic, Jeany Delafiori, Mohammed Shahraz, Corinna Münch, Smiths S. Lueong, Sven T. Liffers, Doris Hellerschmied, Theodore Alexandrov, Jens T. Siveke. Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics [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 4436.

Long-term MNNG exposure promotes gastric carcinogenesis by activating METTL3/m6A/miR1184 axis-mediated epithelial-mesenchymal transition

As the representative item of environmental chemical carcinogen, MNNG was closely associated with the onset of Gastric cancer (GC), while the underlying mechanisms remain largely unknown. Here, we comprehensively analyzed the potential clinical significance of METTL3 in multiple GC patient cohorts. Additionally, we demonstrated that long-term exposure to MNNG elevated METTL3 and EMT marker expression by in vitro and in vivo models. Furthermore, the depletion of METTL3 impacted the proliferation, migration, invasion, and tumorigenesis of MNNG malignant transformation cells and GC cells. By me-RIP sequencing, we identified a panel of vital miRNAs potentially regulated by METTL3 that aberrantly expressed in MNNG-induced GC cells. Mechanistically, we showed that METTL3 meditated miR-1184/TRPM2 axis by regulating the process of miRNA-118. Our results provide novel insights into critical epigenetic molecular events vital to MNNG-induced gastric carcinogenesis. These findings suggest the potential therapeutic targets of METTL3 for GC treatment.

EP06.05-15 STAS and Expression of EMT-markers in Surgically Resected Lung Pleomorphic Carcinomas

EP06.05-15 STAS and Expression of EMT-markers in Surgically Resected Lung Pleomorphic Carcinomas