EMT-like Phenotype Research Articles

TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis.

The heterogeneity of epithelial-to-mesenchymal transition (EMT) programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers. In these cells, TGF-β induces growth inhibition accompanied by a full EMT response that subsequently transitions into an atypical mesenchymal state of round morphology and lacking actin stress fibers. TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy.

IL-1β-activated PI3K/AKT and MEK/ERK pathways coordinately promote induction of partial epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT) is a cellular process in embryonic development, wound healing, organ fibrosis, and cancer metastasis. Previously, we and others have reported that proinflammatory cytokine interleukin-1β (IL-1β) induces EMT. However, the exact mechanisms, especially the signal transduction pathways, underlying IL-1β-mediated EMT are not yet completely understood. Here, we found that IL-1β stimulation leads to the partial EMT-like phenotype in human lung epithelial A549 cells, including the gain of mesenchymal marker (vimentin) and high migratory potential, without the complete loss of epithelial marker (E-cadherin). IL-1β-mediated partial EMT induction was repressed by PI3K inhibitor LY294002, indicating that the PI3K/AKT pathway plays a significant role in the induction. In addition, ERK1/2 inhibitor FR180204 markedly inhibited the IL-1β-mediated partial EMT induction, demonstrating that the MEK/ERK pathway was also involved in the induction. Furthermore, we found that the activation of the PI3K/AKT and MEK/ERK pathways occurred downstream of the epidermal growth factor receptor (EGFR) pathway and the IL-1 receptor (IL-1R) pathway, respectively. Our findings suggest that the PI3K/AKT and MEK/ERK pathways coordinately promote the IL-1β-mediated partial EMT induction. The inhibition of not one but both pathways is expected yield clinical benefits by preventing partial EMT-related disorders such as organ fibrosis and cancer metastasis.

Unveiling epithelial plasticity regulation in lung cancer: Exploring the cross-talk among Tks4 scaffold protein partners.

The epithelial-to-mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein, Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer. In this study, we used CRISPR/Cas9 method to knockout (KO) Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectrometry on five different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

SOX4 reversibly induces phenotypic changes by suppressing the epithelial marker genes in human keratinocytes.

SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.

Tracking the EMT-like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes

Melanoma continues to be a leading cause of mortality among skin cancers. Despite advancements in targeted therapy, patients frequently develop resistance, leading to disease progression within a year. This resistance may result from the epithelial-to-mesenchymal transition (EMT)-like phenotype switching of melanoma cells. Tracking EMT-related phenotypic changes on extracellular vesicles (EVs) has potential to inform early about response to targeted therapy and melanoma progression. However, the knowledge on protein biomarkers carried by melanoma EVs involved in the EMT-like process remains unexplored. Herein, we present a biosensor integrating surface-enhanced Raman scattering and alternating current electrohydrodynamics-induced nanomixing enhancement, for sensitive detection of EMT-associated biomarkers on EV surfaces during targeted therapy. This biosensor successfully tracks the EMT-like phenotype switching in melanoma cell lines treated with mitogen-activated protein kinase inhibitor (MAPKi). Longitudinal monitoring of patients who receive MAPKi therapy and develop resistance, our biosensor shows its ability to identify the EMT-like phenotype switching on circulating EVs. This ability potentially can be leveraged to predict the development of resistance to targeted therapy, allowing for timely intervention and personalized treatment strategies.

Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells

BackgroundTrastuzumab is the only approved target agent for the first-line treatment of human epidermal growth factor receptor-2 (HER-2) positive gastric cancer; however, trastuzumab resistance is a major problem in clinical practice. To comprehend the mechanism of trastuzumab resistance, we focused on the Wnt/β-catenin signaling pathway and its influence on the phenotypes and behavior of trastuzumab-resistant gastric cancer cells.MethodsTrastuzumab-resistant NCI-N87R cells were established in vitro from the human gastric cancer cell line NCI-N87 by dose-escalating repeated trastuzumab treatment. We investigated the phenotypes of NCI-N87R cells, including Wnt signaling pathway activity. Gastric cancer organoid cells were incubated with complete medium and Wnt3a-depletion medium, and their resistance to trastuzumab was compared.ResultsNCI-N87R exhibited stemness and epithelial-mesenchymal transition (EMT)-like phenotypes, along with decreased levels of the epithelial marker E-cadherin and increased levels of the mesenchymal markers Vimentin and Snail along with an increased Wnt signaling pathway activity. When gastric cancer cells were incubated in Wnt3a-conditioned medium. Wnt signaling pathway activity and resistance to trastuzumab increased. Gastric cancer patient-derived organoids incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than those incubated in complete medium.ConclusionsTrastuzumab-resistant gastric cancer cells exhibited EMT-like phenotype, and trastuzumab resistance was promoted by the Wnt/β-catenin signaling pathway. The Wnt/β-catenin pathway is a key signaling pathway for trastuzumab resistance in gastric cancer cells.

Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma.

Ezrin-binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me-EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial-mesenchymal transition (EMT)-like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)-like properties. Shotgun proteomics analysis of proteins that co-immunoprecipitated with EBP50 revealed that Me-EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me-EBP50 knockout, and blebbistatin treatment potentiated the effects of Me-EBP50 knockout. In OCCC cells from clinical samples, Me-EBP50 and MYH9 were co-localized at the apical plasma membrane. Patients with a combination of Me-EBP50-high and MYH9-high scores had the best prognosis for overall and progression-free survival. Our data suggest that Me-EBP50 has tumor-suppressive effects through the establishment and maintenance of epithelial polarization. In contrast, loss of Me-EBP50 expression induces EMT-like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC-like properties, which in turn promote OCCC progression.

Differential miRNA expression of hypoxic MCF7 and PANC-1 cells.

Hypoxia plays a critical role in the tumor microenvironment by affecting cellular proliferation, metabolism, apoptosis, DNA repair, and chemoresistance. Since hypoxia provokes a distinct shift of microRNA, it is important to illustrate the relative contribution of each hypoxamiR to cancer progression. The present study aims to shed light on the hypoxamiRs that are involved in pancreatic and breast cancer progression to highlight novel targets for the development of new therapies. For 20 cycles, MCF7 breast cancer cells and PANC-1 pancreatic cancer cells were subjected to chronic cyclic hypoxia, which consisted of 72 hours of hypoxia followed by 24 hours of reoxygenation. After 10 and 20 cycles of hypoxia, miRNA expression alterations were profiled using RT-PCR array and further analyzed using a visual analytics platform. The MTT cell proliferation assay was used to determine hypoxic cells' chemoresistance to doxorubicin. Under chronic cyclic hypoxia, hypoxic PANC-1 cells have a comparable doubling time with their normoxic counterparts, whereas hypoxic MCF7 cells show a massive increase in doubling time when compared to their normoxic counterparts. Both hypoxic cell lines developed EMT-like phenotypes as well as doxorubicin resistance. According to the findings of miRNet, 6 and 10 miRNAs were shown to play an important role in enriching six hallmarks of pancreatic cancer in the 10th and 20th cycles of hypoxia, respectively, while 7 and 11 miRNAs were shown to play an important role in enriching the four hallmarks of breast cancer in the 10th and 20th cycles of hypoxia, respectively. miR-221, miR-21, miR-155, and miR-34 were found to be involved in the potentiation of hypoxic PANC-1 hallmarks at both the 10th and 20th cycles, while miR-93, miR-20a, miR-15, and miR-17 were found to be involved in the potentiation of hypoxic MCF7 hallmarks at both the 10th and 20th cycles. This variation in miRNA expression was also connected to the emergence of an EMT-like phenotype, alterations in proliferation rates, and doxorubicin resistance. The chemosensitivity results revealed that chronic cyclic hypoxia is critical in the formation of chemoresistant phenotypes in pancreatic and breast cancer cells. miR-181a and let-7e expression disparities in PANC1, as well as miR-93, miR-34, and miR-27 expression disparities in MCF7, may be associated with the formation of chemoresistant MCF7 and PANC-1 cells following 20 cycles of chronic cyclic hypoxia. Indeed, further research is needed since the particular mechanisms that govern these processes are unknown.

Dermokine mutations contribute to epithelial-mesenchymal transition and advanced melanoma through ERK/MAPK pathways.

To discover vulnerabilities associated with dermokine (DMKN) as a new trigger of the epithelial-mesenchymal transition (EMT) -driven melanoma, we undertook a genome-wide genetic screening using transgenic. Here, we showed that DMKN expression could be constitutively increased in human malignant melanoma (MM) and that this correlates with poor overall survival in melanoma patients, especially in BRAF-mutated MM samples. Furthermore, in vitro, knockdown of DMKN inhibited the cell proliferation, migration, invasion, and apoptosis of MM cancer cells by the activation of ERK/MAPK signaling pathways and regulator of STAT3 in downstream molecular. By interrogating the in vitro melanoma dataset and characterization of advanced melanoma samples, we found that DMKN downregulated the EMT-like transcriptional program by disrupting EMT cortical actin, increasing the expression of epithelial markers, and decreasing the expression of mesenchymal markers. In addition, whole exome sequencing was presented with p.E69D and p.V91A DMKN mutations as a novel somatic loss of function mutations in those patients. Moreover, our purposeful proof-of-principle modeled the interaction of ERK with p.E69D and p.V91A DMKN mutations in the ERK-MAPK kinas signaling that may be naturally associated with triggering the EMT during melanomagenesis. Altogether, these findings provide preclinical evidence for the role of DMKN in shaping the EMT-like melanoma phenotype and introduced DMKN as a new exceptional responder for personalized MM therapy.

Interferon-γ inducible protein 30 promotes the epithelial-mesenchymal transition-like phenotype and chemoresistance by activating EGFR/AKT/GSK3β/β-catenin pathway in glioma.

Previous studies have indicated that IFI30 plays a protective role in human cancers. However, its potential roles in regulating glioma development are not fully understood. Public datasets, immunohistochemistry, and western blotting (WB) were used to evaluate the expression of IFI30 in glioma. The potential functions and mechanisms of IFI30 were examined by public dataset analysis; quantitative real-time PCR; WB; limiting dilution analysis; xenograft tumor assays; CCK-8, colony formation, wound healing, and transwell assays; and immunofluorescence microscopy and flow cytometry. IFI30 was significantly upregulated in glioma tissues and cell lines compared with corresponding controls, and the expression level of IFI30 was positively associated with tumor grade. Functionally, both in vivo and in vitro evidence showed that IFI30 regulated the migration and invasion of glioma cells. Mechanistically, we found that IFI30 dramatically promoted the epithelial-mesenchymal transition (EMT)-like process by activating the EGFR/AKT/GSK3β/β-catenin pathway. In addition, IFI30 regulated the chemoresistance of glioma cells to temozolomide directly via the expression of the transcription factor Slug, a key regulator of the EMT-like process. The present study suggests that IFI30 is a regulator of the EMT-like phenotype and acts not only as a prognostic marker but also as a potential therapeutic target for temozolomide-resistant glioma.

Bone marrow mesenchymal/fibroblastic stromal cells induce a distinctive EMT-like phenotype in AML cells

The development of epithelial-to-mesenchymal transition (EMT) like features is emerging as a critical factor involved in the pathogenesis of acute myeloid leukaemia (AML). However, the extracellular signals and the signalling pathways in AML that may regulate EMT remain largely unstudied. We found that the bone marrow (BM) mesenchymal/fibroblastic cell line HS5 induces an EMT-like migratory phenotype in AML cells. AML cells underwent a strong increase of vimentin (VIM) levels that was not mirrored to the same extent by changes of expression of the other EMT core proteins SNAI1 and SNAI2. We validated these particular pattern of co-expression of core-EMT markers in AML cells by performing an in silico analysis using datasets of human tumours. Our data showed that in AML the expression levels of VIM does not completely correlate with the co-expression of core EMT markers observed in epithelial tumours. We also found that vs epithelial tumours, AML cells display a distinct patterns of co-expression of VIM and the actin binding and adhesion regulatory proteins that regulate F-actin dynamics and integrin-mediated adhesions involved in the invasive migration in cells undergoing EMT. We conclude that the BM stroma induces an EMT related pattern of migration in AML cells in a process involving a distinctive regulation of EMT markers and of regulators of cell adhesion and actin dynamics that should be further investigated. Understanding the tumour specific signalling pathways associated with the EMT process may contribute to the development of new tailored therapies for AML as well as in different types of cancers.

Altered expression of anti-apoptotic protein Api5 affects breast tumorigenesis

BackgroundApoptosis or programmed cell death plays a vital role in maintaining homeostasis and, therefore, is a tightly regulated process. Deregulation of apoptosis signalling can favour carcinogenesis. Apoptosis inhibitor 5 (Api5), an inhibitor of apoptosis, is upregulated in cancers. Interestingly, Api5 is shown to regulate both apoptosis and cell proliferation. To address the precise functional significance of Api5 in carcinogenesis here we investigate the role of Api5 in breast carcinogenesis.MethodsInitially, we carried out in silico analyses using TCGA and GENT2 datasets to understand expression pattern of API5 in breast cancer patients followed by investigating the protein expression in Indian breast cancer patient samples. To investigate the functional importance of Api5 in breast carcinogenesis, we utilised MCF10A 3D breast acinar cultures and spheroid cultures of malignant breast cells with altered Api5 expression. Various phenotypic and molecular changes induced by altered Api5 expression were studied using these 3D culture models. Furthermore, in vivo tumorigenicity studies were used to confirm the importance of Api5 in breast carcinogenesis.ResultsIn-silico analysis revealed elevated levels of Api5 transcript in breast cancer patients which correlated with poor prognosis. Overexpression of Api5 in non-tumorigenic breast acinar cultures resulted in increased proliferation and cells exhibited a partial EMT-like phenotype with higher migratory potential and disruption in cell polarity. Furthermore, during acini development, the influence of Api5 is mediated via the combined action of FGF2 activated PDK1-Akt/cMYC signalling and Ras-ERK pathways. Conversely, Api5 knock-down downregulated FGF2 signalling leading to reduced proliferation and diminished in vivo tumorigenic potential of the breast cancer cells.ConclusionTaken together, our study identifies Api5 as a central player involved in regulating multiple events during breast carcinogenesis including proliferation, and apoptosis through deregulation of FGF2 signalling pathway.

Cellular Transcriptomics of Carboplatin Resistance in a Metastatic Canine Osteosarcoma Cell Line

Osteosarcoma prognosis has remained unchanged for the past three decades. In both humans and canines, treatment is limited to excision, radiation, and chemotherapy. Chemoresistance is the primary cause of treatment failure, and the trajectory of tumor evolution while under selective pressure from treatment is thought to be the major contributing factor in both species. We sought to understand the nature of platinum-based chemotherapy resistance by investigating cells that were subjected to repeated treatment and recovery cycles with increased carboplatin concentrations. Three HMPOS-derived cell lines, two resistant and one naïve, underwent single-cell RNA sequencing to examine transcriptomic perturbation and identify pathways leading to resistance and phenotypic changes. We identified the mechanisms of acquired chemoresistance and inferred the induced cellular trajectory that evolved with repeated exposure. The gene expression patterns indicated that acquired chemoresistance was strongly associated with a process similar to epithelial-mesenchymal transition (EMT), a phenomenon associated with the acquisition of migratory and invasive properties associated with metastatic disease. We conclude that the observed trajectory of tumor adaptability is directly correlated with chemoresistance and the phase of the EMT-like phenotype is directly affected by the level of chemoresistance. We infer that the EMT-like phenotype is a critical component of tumor evolution under treatment pressure and is vital to understanding the mechanisms of chemoresistance and to improving osteosarcoma prognosis.

Loganic acid regulates the transition between epithelial and mesenchymal-like phenotypes by alleviating MnSOD expression in hepatocellular carcinoma cells

AimsCancer metastasis is the major cause of cancer-related deaths. There are few prior studies reported on molecules targeting C-X-C chemokine receptor (CXCR) family and manganese superoxide dismutase (MnSOD). CXCRs are known to involve in angiogenesis, metastasis, cell survival and MnSOD is reported to be related in Epithelial–mesenchymal transition (EMT). Main methodsCell viability and cell proliferation were measured by MTT and BrdU assay. Protein expression level of CXCR4/7, MMP-2/9, MnSOD, and EMT markers were evaluated by Western blot analysis. mRNA levels of Snail and Occludin were analyzed by Real-time RT-qPCR. Expression of EMT markers in cells was observed by immunocytochemistry. Cell invasion and migrations were evaluated by wound healing assay and boyden chamber assay. Key findingsWe noticed that LGA abolished proliferation, invasive ability, and cellular migration. LGA down-regulated the protein levels of mesenchymal markers such as Twist, Snail, Fibronectin, and Vimentin in CXCL12-treated HCC cells. It also suppressed the gelatinolytic activity of MMP-9/2. The amplification of MnSOD increased EMT-like phenotypes but with LGA treatment, these phenotypes were markedly attenuated. The overexpression of MnSOD increased the ROS levels significantly but ROS levels were decreased upon exposure to LGA and deletion of MnSOD suppressed the levels of various mesenchymal proteins. SignificanceLGA could function as a novel anti-metastatic agent by suppressing metastasis and EMT process via attenuation of MnSOD expression in hepatocellular carcinoma cells.

Abstract A028: ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism

Abstract Introduction: Epithelial to Mesenchymal Transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therefore, therapeutic targeting of EMT-like phenotypes in cancer cells may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Recently, GPX4 inhibitors (GPX4i) became of interest in this field as they were identified as potent and selective compounds for cells in a mesenchymal state through induction of lipid peroxidation induced cell death also known as ferroptosis. The sensitivity to these inhibitors was also shown to be dependent on the master EMT regulator, ZEB1. However, the link between ZEB1 expression and ferroptosis susceptibility remains unclear. Methodology: We used PC-3E and TEM 4-18 cell lines, originally isolated from the PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer, as our models of epithelial and EMT-like phenotypes respectively. To better understand the role of ZEB1 in ferroptosis susceptibility, we used ZEB1 KO TEM 4-18 cells as well as a panel of cancer cell lines that cover a broad spectrum of epithelial and EMT-like phenotypes. After evaluating the sensitivity to GPX4i in these cell lines, we examined different parameters affecting the lipid peroxidation pathway. We particularly focused on the regulation of iron metabolism by ZEB1. Results: The three GPX4i tested (ML210, ML162 and RSL3) displayed a greater toxicity against TEM 4-18 cells. Inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1) completely reversed the effect of GPX4i indicating that ferroptosis was indeed triggered by GPX4i and responsible for their toxicity. Sensitivity to GPX4i correlated with ZEB1 expression in our panel of cancer cell lines and ZEB1 KO reversed toxicity of GPX4i in TEM 4-18 cells. Sensitivity to GPX4i correlated with a slightly higher labile iron pool in ZEB1 expressing cells. Expression of proteins with a previously established connection to iron metabolism such as Ferritin Light Chain (FtL), Ferritin Heavy Chain (FtH), Transferrin Receptor (TfR) and Prominin-2 (PROM2) were also dysregulated between resistant and sensitive cells. In particular, the ratio FtH:FtL and expression of PROM2 was positively correlated with resistance to GPX4i. In addition, using the Cancer Cell Line Encyclopedia (CCLE) database, we found that ZEB1 expression negatively correlated with PROM2 and Iron Responsive Element Binding Protein-2 (IREB2) suggesting these genes could be transcriptional targets of ZEB1. Conclusions: We validated GPX4i as potent EMT selective cytotoxic compounds and confirmed that the sensitivity to these inhibitors depends on ZEB1 expression. However, the mechanisms leading to sensitivity to GPX4i and how they connect to the EMT program have yet to be identified. As these mechanisms might be diverse, we hypothesize that ZEB1 regulates iron metabolism which is critical for ferroptosis induction. Citation Format: Marion Vanneste, Michael Darrin Henry, Akansha Jain. ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A028.

448 RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties

Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored.

Abstract C072: Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of only 11% and approximately half of patients present with distant metastasis at the time of diagnosis. PDAC is accompanied by pronounced alterations in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDAC tumor evolution. The role of immune modulators in driving metastatic potential in PDAC is poorly understood. We find that low expression levels of cytokine IL-23 in patients with PDAC correlate with poor differentiation status of these tumors, and thus predict poor outcomes. Consistent with this observation, we have found that a loss of host-derived IL-23 in a mouse model of pancreatic cancer promotes an EMT-like phenotype. The tumor morphology is altered and appears to be less differentiated. Additionally, we see a significant increase in the number of tumor cells expressing Zeb1 and a decrease in E-cadherin expression. We also find an increase in metastatic capacity in the absence of IL-23 in the tumor stroma. Single cell RNA sequencing of tumors injected orthotopically into IL-23 knockout mice reveals clusters of tumor cells with increases in EMT-associated genes such as Zeb1 and Vimentin and concurrent decreases in gastric identity genes. Overall, our findings indicate that IL-23 plays a role in suppressing an EMT-like phenotype in pancreatic cancer with one mechanism being through regulation of gastric lineage genes. Citation Format: Whitney Bell, Nancy Kren, Yan Wang, Yuliya Pylayeva-Gupta. Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C072.

Quercetin Suppresses Human Glioblastoma Migration and Invasion via GSK3β/β-catenin/ZEB1 Signaling Pathway.

High invasiveness is a biological and clinical characteristic of glioblastoma and predicts poor prognosis of patients. Quercetin, a natural flavonoid compound, exhibits anticancer activity. However, we have a limited understanding of the possible underlying mechanism of quercetin in glioblastoma. In this study, we investigated the anticancer effect of quercetin in human glioblastoma cells. Our results showed that quercetin markedly suppressed the viability of glioblastoma cells in vitro and in vivo, and significantly inhibited glioblastoma cell migration and invasion. Moreover, quercetin reversed EMT-like mesenchymal phenotype and reduced the expression levels of EMT-related markers. Furthermore, we found that quercetin suppressed GSK-3β/β-catenin/ZEB1 signaling in glioblastoma. Taken together, our results demonstrate that quercetin inhibited migration and invasion of human glioma cells by suppressing GSK3β/β-catenin/ZEB1 signaling. Our study provides evidence that quercetin is a promising therapeutic natural compound to treat glioblastoma.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

Investigating the Role of CTCs with Stem/EMT-like Features in Metastatic Breast Cancer Patients Treated with Eribulin Mesylate.

Simple SummaryEribulin mesylate, an anti-mitotic drug used for the treatment of metastatic breast cancer (BC), exhibits significant effects on cancer cell migration, invasion, and metastatic seeding in experimental models. Interestingly, eribulin treatment has been shown to target the cancer stem cell (CSC) subsets in vitro and reverse the epithelial-to-mesenchymal transition (EMT) state of BC cells. In the current study, circulating tumor cells (CTCs) identified in the peripheral blood of patients with metastatic BC were analyzed at different time points during eribulin treatment and on disease progression. The results contribute new data on the mechanisms of resistance to eribulin mesylate and the prognostic relevance of CTC analyses for eribulin-treated metastatic BC.We herein aimed to assess the effect of eribulin mesylate on the cancer stem cell (CSC)/EMT-like phenotype of CTCs, and to investigate the prognostic role of CTC detection and monitoring for eribulin-treated BC patients. Peripheral blood was obtained at baseline (n = 42 patients) and 8 days after treatment initiation (C1D8: n = 22), and on disease progression (PD: n = 26). PBMCs cytospins were immunofluorescently stained for Cytokeratins/ALDH1/TWIST1/DAPI and analyzed via Ariol microscopy. CTCs were detected in 33.3%, 27.3%, and 23.1% of patients at baseline, C1D8, and PD, respectively. Accordingly, partial-EMT+ CTCs represented 61.3%, 0%, and 37.5% of total CTCs, whereas the CSC-like phenotype was consistently expressed by 87.5%, 75%, and 91.7% of CTCs at the respective time points. Interestingly, the CSC+/partial-EMT+ subset prevailed at baseline, but it was eradicated on C1D8 and resurged again during PD. CTC detection at baseline was associated with reduced PFS (p = 0.007) and OS (p = 0.005), and was an independent risk factor for death (HR: 3.779, p = 0.001; multivariate analysis). The CSC+/partial-EMT+ CTCs emerged as the only subset with adverse prognostic significance, while CTC monitoring during eribulin therapy improved the prediction of disease progression. These results indicate that resistant CTC subsets persevere eribulin treatment and highlight the prognostic implications of CTC analyses for eribulin-treated BC patients.