Acquisition Of Mesenchymal Features Research Articles

Ferroptosis and EMT resistance in cancer: a comprehensive review of the interplay.

Ferroptosis differs from traditional cell death mechanisms like apoptosis, necrosis, and autophagy, primarily due to its reliance on iron metabolism and the loss of glutathione peroxidase activity, leading to lipid peroxidation and cell death. The dysregulation of iron metabolism is a hallmark of various cancers, contributing to tumor progression, metastasis, and notably, drug resistance. The acquisition of mesenchymal characteristics by epithelial cells is known as Epithelial-Mesenchymal Transition (EMT), a biological process intricately linked to cancer development, promoting traits such as invasiveness, metastasis, and resistance to therapeutic interventions. EMT plays a pivotal role in cancer progression and contributes significantly to the complex dynamics of carcinogenesis. Research findings indicate that mesenchymal cancer cells exhibit greater susceptibility to ferroptosis compared to their epithelial counterparts. The induction of ferroptosis becomes more effective in eliminating drug-resistant cancer cells during the process of EMT. The interplay between ferroptosis and EMT, a process where epithelial cells transform into mobile mesenchymal cells, is crucial in understanding cancer progression. EMT is associated with increased cancer metastasis and drug resistance. The review delves into how ferroptosis and EMT influence each other, highlighting the role of key proteins like GPX4, which protects against lipid peroxidation, and its inhibition can induce ferroptosis. Conversely, increased GPX4 expression is linked to heightened resistance to ferroptosis in cancer cells. Moreover, the review discusses the implications of EMT-induced transcription factors such as Snail, Zeb1, and Twist in modulating the sensitivity of tumor cells to ferroptosis, thereby affecting drug resistance and cancer treatment outcomes. Targeting the ferroptosis pathway offers a promising therapeutic strategy, particularly for tumors resistant to conventional treatments. The induction of ferroptosis in these cells could potentially overcome drug resistance. However, translating these findings into clinical practice presents challenges, including understanding the precise mechanisms of ferroptosis induction, identifying predictive biomarkers, and optimizing combination therapies. The review underscores the need for further research to unravel the complex interactions between ferroptosis, EMT, and drug resistance in cancer. This could lead to the development of more effective, targeted cancer treatments, particularly for drug-resistant tumors, offering new hope in cancer therapeutics.

Dual role of CASP8AP2/FLASH in regulating epithelial-to-mesenchymal transition plasticity (EMP)

BackgroundEpithelial-to-mesenchymal transition (EMT) is a developmental program that consists of the loss of epithelial features concomitant with the acquisition of mesenchymal features. Activation of EMT in cancer facilitates the acquisition of aggressive traits and cancer invasion. EMT plasticity (EMP), the dynamic transition between multiple hybrid states in which cancer cells display both epithelial and mesenchymal markers, confers survival advantages for cancer cells in constantly changing environments during metastasis. MethodsRNAseq analysis was performed to assess genome-wide transcriptional changes in cancer cells depleted for histone regulators FLASH, NPAT, and SLBP. Quantitative PCR and Western blot were used for the detection of mRNA and protein levels. Computational analysis was performed on distinct sets of genes to determine the epithelial and mesenchymal score in cancer cells and to correlate FLASH expression with EMT markers in the CCLE collection. ResultsWe demonstrate that loss of FLASH in cancer cells gives rise to a hybrid E/M phenotype with high epithelial scores even in the presence of TGFβ, as determined by computational methods using expression of predetermined sets of epithelial and mesenchymal genes. Multiple genes involved in cell-cell junction formation are similarly specifically upregulated in FLASH-depleted cells, suggesting that FLASH acts as a repressor of the epithelial phenotype. Further, FLASH expression in cancer lines is inversely correlated with the epithelial score. Nonetheless, subsets of mesenchymal markers were distinctly up-regulated in FLASH, NPAT, or SLBP-depleted cells. ConclusionsThe ZEB1low/SNAILhigh/E-cadherinhigh phenotype described in FLASH-depleted cancer cells is driving a hybrid E/M phenotype in which epithelial and mesenchymal markers coexist.

Involvement of Epithelial-Mesenchymal Transition (EMT) in Autoimmune Diseases.

Epithelial-mesenchymal transition (EMT) is a complex reversible biological process characterized by the loss of epithelial features and the acquisition of mesenchymal features. EMT was initially described in developmental processes and was further associated with pathological conditions including metastatic cascade arising in neoplastic progression and organ fibrosis. Fibrosis is delineated by an excessive number of myofibroblasts, resulting in exuberant production of extracellular matrix (ECM) proteins, thereby compromising organ function and ultimately leading to its failure. It is now well acknowledged that a significant number of myofibroblasts result from the conversion of epithelial cells via EMT. Over the past two decades, evidence has accrued linking fibrosis to many chronic autoimmune and inflammatory diseases, including systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and inflammatory bowel diseases (IBD). In addition, chronic inflammatory states observed in most autoimmune and inflammatory diseases can act as a potent trigger of EMT, leading to the development of a pathological fibrotic state. In the present review, we aim to describe the current state of knowledge regarding the contribution of EMT to the pathophysiological processes of various rheumatic conditions.

Morphological features of epithelial-mesenchymal transformation and its effect on tumor progression of breast cancer

Introduction. Breast cancer is in the first place in the structure of morbidity among all malignant neoplasms in women. The prognosis of the disease depends on the tumor degree, including the presence of epithelial-mesenchymal transformation (EMT), the degree of invasion, the proliferative index, the preservation or absence of estrogen, progesterone, and epidermal growth factor receptors.Aim: To study the immunohistochemical and morphological characteristics of the epithelial-mesenchymal transformation of breast cancer.Material and Methods. Immunohistochemical study with antibodies to AE1/AE3, HMW, CK18, Snail, HER2/neu, E-cadherin, Vimentin, α-SMA, CD34, Ki-67 and p63 was performed in 60 patients of different age with breast cancer. Native preparations were stained with picrofuchsin according to van Gieson Alcian blue. Inflammatory infiltrate cells were examined for antibodies CD45, CD3, CD4, CD8, CD20, CD68.Results. In ductal carcinoma, positive expression for estrogen and progesterone was found in 82.7% and 86.3%, respectively, the proliferation index ranged before 66,6%, and p-53 was positive in 97%. In lobular cancer, positive expression to estrogen and progesterone was observed in 83.4% and 66.6%, respectively, the index of proliferative activity at the level of 50%, and p-53 was positive in 66.6%. Positive moderate expression of HER-2/neu was determined in 47% of ductal and 50% of lobular cancers. Estrogen plays an important role in the development of invasive breast cancer, leads to tumor progression and contributes to EMT. EMT, in turn, leads to the expression of E-cadherin associated with a worse survival prognosis. EMT indirectly leads to the intensification of angiogenesis, and the presence of a large number of newly formed vessels increases the risk of metastasis. Histochemical methods were used to determine the growth of fibrous tissue around invasively growing cancer complexes. Cells located perifocally looked like fibrobla ts, immunohistochemically moderately expressed Vimentin and weakly expressed pancytokeratin, which proved the tumor nature of the cells and the acquisition of mesenchymal features by them. The inflammatory infiltrate along the periphery of the tumor growth consisted mainly of T- and B-lymphocytes, and around the cancer complexes - of B-lymphocytes and macrophages.Conclusion. The study of the immunohistochemical tumor phenotype will make it possible to prescribe adequate polychemotherapy and determine the prognosis of the course of the disease.

USP7 imparts partial EMT state in colorectal cancer by stabilizing the RNA helicase DDX3X and augmenting Wnt/β-catenin signaling

Epithelial mesenchymal transition (EMT) is a fundamental and highly regulated process that is normally observed during embryonic development and tissue repair but is deregulated during advanced cancer. Classically, through the process of EMT, cancer cells gradually transition from a predominantly epithelial phenotype to a more invasive mesenchymal phenotype. Increasing studies have, however, brought into light the existence of unique intermediary states in EMT, often referred to as partial EMT states. Through our studies we have found the deubiquitinase USP7 to be strongly associated with the development of such a partial EMT state in colon cancer cells, characterized by the acquisition of mesenchymal characteristics but without the reduction in epithelial markers. We found USP7 to be overexpressed in colon adenocarcinomas and to be closely associated with advancing tumor stage. We found that functional inhibition or knockdown of USP7 is associated with a marked reduction in mesenchymal markers and in overall migration potential of cancer cells. Starting off with a proteomics-based approach we were able to identify and later on verify the DEAD box RNA helicase DDX3X to be an interacting partner of USP7. We then went on to show that USP7, through the stabilization of DDX3X, augments Wnt/β-catenin signaling, which has previously been shown to be greatly associated with colorectal cancer cell invasiveness. Our results indicate USP7 as a novel key player in establishing a partial mesenchymal phenotype in colorectal cancer.

Morphological features of epithelial-mesenchymal transformation and its effect on tumor progression of breast cancer

Introduction. Breast cancer is in the first place in the structure of morbidity among all malignant neoplasms in women. The prognosis of the disease depends on the tumor degree, including the presence of epithelial-mesenchymal transformation (EMT), the degree of invasion, the proliferative index, the preservation or absence of estrogen, progesterone, and epidermal growth factor receptors.Aim. To study the immunohistochemical and morphological characteristics of the epithelial-mesenchymal transformation of breast cancer.Material and Methods. Immunohistochemical study with antibodies to AE1/AE3, HMW, CK18, Snail, HER2/neu, E-cadherin, Vimentin, α-SMA, CD34, Ki-67 and p63 was performed in 60 patients of different age with breast cancer. Native preparations were stained with picrofuchsin according to van Gieson Alcian blue. Inflammatory infiltrate cells were examined for antibodies CD45, CD3, CD4, CD8, CD20, CD68.Results. In ductal carcinoma, positive expression for estrogen and progesterone was found in 82.7% and 86.3%, respectively, the proliferation index ranged before 66,6 %, and p-53 was positive in 97%. In lobular cancer, positive expression to estrogen and progesterone was observed in 83.4% and 66.6%, respectively, the index of proliferative activity at the level of 50 %, and p-53 was positive in 66.6%. Positive moderate expression of HER-2/neu was determined in 47% of ductal and 50% of lobular cancers. Estrogen plays an important role in the development of invasive breast cancer, leads to tumor progression and contributes to EMT. EMT, in turn, leads to the expression of E-cadherin associated with a worse survival prognosis. EMT indirectly leads to the intensification of angiogenesis, and the presence of a large number of newly formed vessels increases the risk of metastasis. Histochemical methods were used to determine the growth of fibrous tissue around invasively growing cancer complexes. Cells located perifocally looked like fibroblasts, immunohistochemically moderately expressed Vimentin and weakly expressed pancytokeratin, which proved the tumor nature of the cells and the acquisition of mesenchymal features by them. The inflammatory infiltrate along the periphery of the tumor growth consisted mainly of T- and B-lymphocytes, and around the cancer complexes - of B-lymphocytes and macrophages.Conclusion. The study of the immunohistochemical tumor phenotype will make it possible to prescribe adequate polychemotherapy and determine the prognosis of the course of the disease.

The Association of RGS2 and Slug in the Androgen-induced Acquisition of Mesenchymal Features of Breast MDA-MB-453 Cancer Cells

ABSTRACT Background Epithelial-mesenchymal transition (EMT) of tumor cells is a prerequisite to cancer cell invasion and metastasis. This process involves a network of molecular alterations. Androgen receptor (AR) plays an important role in the biology of breast cancers, particularly those dependent on AR expression like luminal AR (LAR) breast cancer subtype. We have recently reported that the AR agonist, dihydrotestosterone (DHT), induces a mesenchymal transition of MDA-MB-453 cells, concomitant with transcriptional up-regulation of Slug and regulator of G protein signaling 2 (RGS2). Objective The role of Slug and RGS2 in mediating the DHT-induced effects in these cells was investigated. Methods MDA-MB-453 cells were used as a model system of LAR breast cancer. Immunofluorescence was used to examine cell morphology and protein localization. Protein expression was analyzed by immunoblotting. Protein localization was confirmed by cell fractionation followed by immunoblotting. Protein-protein interaction was confirmed by co-immunoprecipitation followed by immunoblotting. Transwell membranes were used to assess cell migration. Transfection of cells with siRNA molecules that target Slug and RGS2 mRNA was utilized to delineate the modes of action of these two molecules. Results Treatment of MDA-MB-453 cells with DHT induced the expression of both proteins. In addition, AR-Slug, AR-RGS2, and Slug-RGS2 interactions were observed shortly after AR activation. Knocking down Slug abrogated the basal, but not the DHT-induced, cell migration and blocked DHT-induced mesenchymal transition. On the other hand, RGS2 knocked-down cells had an increased level of Slug protein and assumed mesenchymal cell morphology with induced migration, and the addition of DHT further elongated cell morphology and stimulated their migration. Inhibition of AR or β-catenin reverted the RGS2 knocked-down cells to the epithelial phenotype, but only inhibition of AR blocked their DHT-induced migration. Conclusions These results suggest the involvement of RGS2 and Slug in a complex molecular network regulating the DHT-induced mesenchymal features in MDA-MB-453 cells. The study may offer a better understanding of the biological role of AR in breast cancer toward devising AR-based therapeutic strategies.

Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells.

Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand-receptor interactions, which may have important implications in cancer.

Abstract PO-059: Epithelial/mesenchymal identity dictates pancreatic cancer cell metastasis

Abstract Metastatic pancreatic adenocarcinoma (PDAC) is the dominant clinical presentation and highly treatment-resistant. However, not all metastasis is equal with metastatic disease in the lung having improved outcomes over the liver. These clinical differences suggest a therapeutic opportunity and urge analysis of the molecular underpinnings of PDAC metastasis. The acquisition of mesenchymal features by epithelial cancer cells is commonly associated with solid tumor metastasis and has been linked to the pancreatic cancer basal subtype and its association with treatment resistance and poorer outcomes, but its impact on pancreatic cancer metastasis needs further understanding. We explored the impact on metastasis of stabilized epithelial, partial-mesenchymal and mesenchymal cancer cells by generating several genetic mouse models based on the lineage-traced KPC mouse model (KrasLSL-G12D;p53R172H; or p53LSL; PDX1-Cre;EYFPLSL). Using single-cell RNA-sequencing we confirmed the KPC mouse model recapitulates the spectrum of epithelial-mesenchymal phenotypes observed in patients and can be genetically engineered to stabilize specific phenotypes. The stabilization of epithelial phenotypes through the homozygous loss of the mesenchymal-driving transcription factors Snail and Twist (Snai1F/F;Twist1F/F) had no impact on primary tumor progression but increased liver colonization. This increase in liver colonization was supported by a second epithelial-stabilized mouse model based on the loss of Zeb1 (Zeb1F/F). The stabilization of mesenchymal features through the heterozygous or homozygous loss of the epithelial adherin junction E-cadherin (Cdh1F/+ or Cdh1F/F) promoted lung metastasis. Interestingly, epithelial plasticity was still required for efficient lung colonization, but not rare liver metastasis. Additionally, mesenchymal gene expression correlated with an improved patient survival as well as metastatic localization, supporting the clinical observations of improved survival in lung metastasis. Using gene expression analysis of sorted bulk cancer cells and single-cells, migration assays, and multiplexed-immunohistochemistry, we observed that the epithelial/mesenchymal status of the cancer cells dictated different mechanisms for motility and interaction with the immune system. Mesenchymal cancer cells migrate as single-cells and attract fewer T-cells where epithelial cancer cells migrate collectively and have increased immune regulation gene expression. These data suggest the epithelial/mesenchymal status of cancers cells dictate the where and how of metastatic disease and could inform therapeutic interventions. Citation Format: Julienne L. Carstens, Sujuan Yang, Pedro Correa de Sampaio, Xiaofeng Zheng, Souptik Barua, Kathleen M. McAndrews, Arvind Rao, Jared K. Burks, Andrew D. Rhim, Raghu Kalluri. Epithelial/mesenchymal identity dictates pancreatic cancer cell metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-059.

Epithelial to mesenchymal transition and microRNA expression are associated with spindle and apocrine cell morphology in triple-negative breast cancer

Triple negative breast cancers (TNBC) are a morphologically and genetically heterogeneous group of breast cancers with uncertain prediction of biological behavior and response to therapy. Epithelial to mesenchymal transition (EMT) is a dynamic process characterized by loss of typical epithelial phenotype and acquisition of mesenchymal characteristics. Aberrant activation of EMT can aggravate the prognosis of patients with cancer, however, the mechanisms of EMT and role of microRNAs (miRNAs) in EMT activation is still unclear. The aim of our study was to analyze miRNA expression within areas of TNBCs with cellular morphology that may be related to the EMT process and discuss possible associations. Out of all 3953 re-examined breast cancers, 460 breast cancers were diagnosed as TNBC (11.64%). With regard to complete tumor morphology preservation, the tissue samples obtained from core—cut biopsies and influenced by previous neoadjuvant therapy were excluded. We assembled a set of selected 25 cases to determine miRNA expression levels in relation to present focal spindle cell and apocrine cell morphology within individual TNBCs. We used descriptive (histological typing and morphology), morphometric, molecular (microdissection of tumor and non-tumor morphologies, RNA isolation and purification, microchip analysis) and bioinformatic analysis (including pathway analysis). The results were verified by quantitative real-time PCR (RT-qPCR) on an extended set of 70 TNBCs. The majority of TNBCs were represented by high—grade invasive carcinomas of no special type (NST) with medullary features characterized by well-circumscribed tumors with central necrosis or fibrosis and frequent tendency to spindle-cell and/or apocrine cell transformation. Apocrine and spindle cell transformation showed a specific miRNA expression profile in comparison to other tumor parts, in situ carcinoma or non-tumor structures, particularly down-regulated expression of hsa-miRNA-143-3p and hsa-miRNA-205-5p and up-regulated expression of hsa-miR-22-3p, hsa-miRNA-185-5p, and hsa-miR-4443. Apocrine cell tumor morphology further revealed decreased expression of hsa-miR-145-5p and increased expression of additional 14 miRNAs (e.g. hsa-miR-182-5p, hsa-miR-3135b and hsa-miR-4417). Pathway analysis for target genes of these miRNAs revealed several shared biological processes (i.e. Wnt signaling, ErbB signaling, MAPK signaling, endocytosis and axon guidance), which may in part contribute to the EMT and tumor progression. We provide the first miRNA expression profiling of specific tissue morphologies in TNBC. Our results demonstrate a specific miRNA expression profile of apocrine and spindle cell morphology which can exhibit a certain similarity with the EMT process and may also be relevant for prognosis and therapy resistance of TNBC.

Loss of ARID1A Promotes Epithelial-Mesenchymal Transition and Sensitizes Pancreatic Tumors to Proteotoxic Stress.

Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in ARID1A represent one of the most frequent molecular alterations in human cancers. ARID1A mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial-mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both in vitro and in vivo. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in ARID1A associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment. SIGNIFICANCE: This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.

Abstract 5107: DDR1 and MT1-MMP expression levels are major determinants of 3D type I collagen-induced apoptosis in basal-like breast carcinoma

Abstract Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen. Citation Format: Charles Saby, Guillaume Collin, Maha Sinane, Emilie Buache, Laurence Van Gulick, Frédéric Saltel, Erik Maquoi, Hamid Morjani. DDR1 and MT1-MMP expression levels are major determinants of 3D type I collagen-induced apoptosis in basal-like breast carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5107.

The CXCR2-SNAIL Axis: Is this a Novel Anti-Tumor Therapeutical Target for Cancer Cells Undergoing Epithelial-Mesenchimal Transition Process?

The epithelial-mesenchymal transition (EMT) plays an important role in the progression of cancer, metastasis and drug resistance. Several factors are known to mediate EMT-driven drug resistance in cancer cells, among them the tumor microenvironment (TME). This phenomenon has gained attention in the field of cancer biology for its potential contribution to in the progression of carcinomas. It is also known that tumor cells experiencing EMT increases the secretion of specific factors in the TME, including cytokines, chemokines and growth factors, which can play an important role in tumor progression. The main event in EMT is the repression of E-cadherin driven by transcriptional factors including SNAIL, SLUG and ZEB1. Chemokines function as growth factors, activating, through its receptor CXCR2 and transcription factors such as SNAIL, thus inducing the EMT phenotype, contributing to the progression of the disease. Studies have investigated how the acquisition of mesenchymal characteristics could contribute to the development of a tumor microenvironment, and point to a possible link between the CXCR2 pathway and EMT. This review describes the mechanism by which CXCR2 is involved in EMT through SNAIL, contributing to progression of cancer and summarizes new advances in the research of EMTassociated CXCR2.

Abstract B04: Multimodal cancer immunotherapy combining IL-8 inhibition, adenovirus vaccine, IL-15 super agonist, and anti-PD-L1/TGFβRII agent reduces mesenchymalization and enhances anti-tumor efficacy

Abstract Tumor cells utilize the secretion of proinflammatory cytokines and chemokines and the surface expression of inhibitory ligands to promote an immune-suppressive tumor microenvironment that facilitates tumor growth and metastasis. The overexpression of IL-8, TGF-β, PD-L1, the IL-8 receptors CXCR1 and CXCR2, as well as numerous other factors by various solid and hematologic cancers are major obstacles in the promotion of a strong and effective antitumor immune response. Both IL-8 and TGF-β have been identified as key potentiators of tumor cell plasticity and the acquisition of mesenchymal features by epithelial cancer cells, i.e., mesenchymalization, which increases metastatic dissemination. Both IL-8 and TGF-β can also promote tumor angiogenesis, myeloid-derived suppressor cell (MDSC) function in the tumor microenvironment, and resistance to immune-mediated killing. Additionally, tumor cell expression of PD-L1 inhibits effective T-cell responses at the tumor site. Here we report the use of a systematic and rational multimodal cancer immunotherapy approach that combines induction of T cell potentiation through an IL-15 super agonist and adenoviral cancer vaccine with inhibition of three immunosuppressive pathways through blockade of IL-8, TGF-β, and PD-L1. Combination of the novel small-molecule inhibitor SX-682 that irreversibly binds and blocks both CXCR1 and CXCR2 with an adenoviral-based cancer vaccine, N-IL15 (previously designated ALT-803), and the anti-PD-L1/TGFβRII agent, M7824, significantly delayed tumor growth in murine models of breast and colon cancer. Mechanistic studies demonstrated that this multimodal combination therapy was able to markedly increase tumor infiltration with CD4+ and CD8+ T cells, including central memory and effector memory subsets, and to decrease infiltration with Ly6G+/Ly6C- MDSC. Studies with SX-682 as a monotherapy suggest that CXCR1/2 inhibition reduces migration rates of Ly6G+/Ly6C- MDSC (CXCR2+). Studies with models of claudin-low, mesenchymal-enriched, human triple-negative breast cancer demonstrated that CXCR1/2 inhibition was also effective at promoting a switch from a mesenchymal to an epithelial tumor phenotype, while simultaneously enhancing the tumor cells’ susceptibility to immune-mediated cytotoxicity. Our results provide rationale for the future evaluation of this multimodal therapy in the clinic. Citation Format: Lucas Horn, Duane Hamilton, Dean Maeda, John Zebala, John Lee, Shahrooz Rabizadeh, Frank Jones, Patrick Soon-Shiong, Zhen Su, Jeffrey Schlom, Claudia Palena. Multimodal cancer immunotherapy combining IL-8 inhibition, adenovirus vaccine, IL-15 super agonist, and anti-PD-L1/TGFβRII agent reduces mesenchymalization and enhances anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B04.

Abstract B43: Simultaneous inhibition of CXCR1/CXCR2, TGF-β, and PD-L1 overcomes immune resistance driven by tumor plasticity

Abstract The occurrence of phenotypic plasticity in carcinomas, which results in the acquisition of mesenchymal features by epithelial cancer cells, associates with a poor prognosis and has been described as a mechanism of primary resistance and failure to treatment with checkpoint blockade immunotherapy targeting the PD-1/PD-L1 pathway. IL-8 and TGF-β are two of the factors that mediate phenotypic plasticity in epithelial tumors to promote an immune-suppressive microenvironment. Here we report on an immunotherapy approach aimed at overcoming escape mechanisms mediated by IL-8 and TGF-β accumulation in the tumor microenvironment via the use of two clinical-stage agents: SX-682, a dual CXCR1/2 small-molecule inhibitor, with the bifunctional anti-PD-L1/TGFβRII agent, bintrafusp alfa (M7824) that simultaneously blocks PD-L1 while trapping soluble TGF-β. We demonstrate that the concurrent inhibitions of CXCR1/CXCR2, TGF-β, and PD-L1 signaling synergize to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells (G-MDSC), significantly enhancing CD4+ and CD8+ T-cell infiltration and activation in tumors, and leading to improved antitumor activity. This combination approach was effective in tumor models with varying immune cell infiltrates, degrees of plasticity, and responses to immunotherapy. The results of these studies provide the rationale for the future evaluation of this multimodal therapy in the clinic. Citation Format: Lucas A. Horn, Jeffrey Riskin, Heidi A. Hempel, Hanne Lind, Kristen Fousek, Duane H. Hamilton, Kristen K. McCampbell, Jeffrey Schlom, Claudia Palena. Simultaneous inhibition of CXCR1/CXCR2, TGF-β, and PD-L1 overcomes immune resistance driven by tumor plasticity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B43.

Simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 remodels the tumor and its microenvironment to drive antitumor immunity

BackgroundDespite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there...

SIRT6-mediated transcriptional suppression of MALAT1 is a key mechanism for endothelial to mesenchymal transition

BackgroundVascular aging has profound effects on cardiovascular diseases. Endothelial to mesenchymal transition (EndMT) is defined as the acquisition of mesenchymal characteristics by endothelial cells (ECs) and has been found induced in a model of ECs aging. However, whether EndMT occurs during aging in vivo, the functional significance of EndMT on vascular biology and the underlying mechanisms remain unknown. Methods and resultsIn this study, we examined the vascular ECs from young (2 months old) and old (18 months old) mice, and demonstrated that aged ECs underwent EndMT. Moreover, the transwell assay showed that EndMT process was accompanied by increased endothelial permeability. It was found that sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide+ (NAD+)-dependent histone deacetylase, was down-regulated during ECs aging. Knockdown of SIRT6 in young ECs could induce EndMT. Next, we identified five long non-coding RNAs that are enriched in ECs for downstream effector of SIRT6; only metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was significantly up-regulated in aged ECs. Knockdown of SIRT6 could increase MALAT1 levels. Furthermore, the ChIP assay and luciferase reporter gene assay confirmed that SIRT6 bound directly to the promoter region of MALAT1 and suppressed MALAT1 expression. Finally, we demonstrated that MALAT1 mediated aging-induced EndMT through increasing Snail expression. ConclusionOur study provides in vivo evidence that ECs undergo EndMT during vascular aging, which increases endothelial permeability. SIRT6-mediated transcriptional suppression of MALAT1 is a key mechanism for EndMT. Manipulating EndMT may be considered as a new therapeutic strategy for retarding aging-associated vascular diseases.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

Distinct biological characterization of the CD44 and CD90 phenotypes of cancer stem cells in gastric cancer cell lines.

Recent study implicates that gastric cancer stem cells (CSCs) are capable of generating multiple types of cells to promote tumor growth and heterogeneity important for the development of gastric cancer. However, knowledge is limited regarding the expression and characteristics of marker-positive gastric CSCs. Therefore, gastric CSCs from a series of human gastric cancer cell lines (SNU-5, SNU-16, BGC-823, PAMC-82, MKN-45, and NCI-N87) using four putative CSC surface markers (CD44, CD90, CD133, and epithelial-cell adhesion molecule) were investigated the underlying mechanisms regulating such subpopulations. Only SNU-5 and SNU-16 exhibited independent co-expression of CD44+ and CD90+, which exhibited spheroid-colony formation in vitro and tumor formation in immunodeficient mice. Functional studies revealed that CD44+ cells were more invasive compared with CD90+ cells, whereas CD90+ cells exhibited higher levels of proliferation than CD44+ cells. Furthermore, serial xenotransplantation in mice of CD44+/CD90+ cells derived from SNU-5 and SNU-16 revealed rapid growth of CD90+ cells in subcutaneous lesions and a high metastatic capacity of CD44+ cells in the lung. Mechanistic analyses revealed that CD44+ cells underwent epithelial-to-mesenchymal transition (EMT) following acquisition of mesenchymal features, whereas CD90+ cells enhanced the activation of retinoblastoma phosphorylation at Ser780 and oncogenic cell cycle regulators. The expression of CD44 and CD90 in gastric cancer tissues was associated with distant metastasis and the differentiation state of tumors. These results demonstrated that CD44 and CD90 are specific biomarkers capable of identifying and isolating metastatic and tumorigenic CSCs through their ability to regulate EMT and the cell cycle in gastric cancer cell lines.

DDR1 and MT1-MMP Expression Levels Are Determinant for Triggering BIK-Mediated Apoptosis by 3D Type I Collagen Matrix in Invasive Basal-Like Breast Carcinoma Cells.

Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen.