Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

ZEB1-mediated fibroblast polarization controls inflammation and sensitivity to immunotherapy in colorectal cancer.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

Zeb1 maintains long-term adult hematopoietic stem cell function and extramedullary hematopoiesis.

Emerging evidence implicates the epithelial-mesenchymal transition transcription factor Zeb1 as a critical regulator of hematopoietic stem cell (HSC) differentiation. Whether Zeb1 regulates long-term maintenance of HSC function remains an open question. Using an inducible Mx-1-Cre mouse model that deletes conditional Zeb1 alleles in the adult hematopoietic system, we found that mice engineered to be deficient in Zeb1 for 32 weeks displayed expanded immunophenotypically defined adult HSCs and multipotent progenitors associated with increased abundance of lineage-biased/balanced HSC subsets and augmented cell survival characteristics. During hematopoietic differentiation, persistent Zeb1 loss increased B cells in the bone marrow and spleen and decreased monocyte generation in the peripheral blood. In competitive transplantation experiments, we found that HSCs from adult mice with long-term Zeb1 deletion displayed a cell autonomous defect in multilineage differentiation capacity. Long-term Zeb1 loss perturbed extramedullary hematopoiesis characterized by increased splenic weight and a paradoxical reduction in splenic cellularity that was accompanied by HSC exhaustion, lineage-specific defects, and an accumulation of aberrant, preleukemic like c-kit+CD16/32+ progenitors. Loss of Zeb1 for up to 42 weeks can lead to progressive splenomegaly and an accumulation of Gr-1+Mac-1+ cells, further supporting the notion that long-term expression of Zeb1 suppresses preleukemic activity. Thus, sustained Zeb1 deletion disrupts HSC functionality in vivo and impairs regulation of extramedullary hematopoiesis with potential implications for tumor suppressor functions of Zeb1 in myeloid neoplasms.

Twenty years of EMT: A state of the field from TEMTIA X.

TEMTIA X, the tenth symposium organized by the EMT international Association (TEMTIA) took place in Paris on November 7th-10th, 2022. Similarly to the previous meetings, it reviewed most recent aspects of the epithelial-mesenchymal transition, a cellular process involved during distinct stages of development, but also during wound healing and fibrosis to some level. EMT steps are likewise typically described with various extents during tumor cell progression and metastasis. The meeting emphasized the intermediate stages involved in the process and their potential physiological or pathological importance, taking advantage of the expansion of molecular methods at single cell level. It also introduced new descriptions of EMT occurrences during early embryogenesis. In addition, sessions explored how EMT reflects cell metabolism and how the process can mingle with immune response, particularly during tumor progression, providing new targets, that were discussed, among others, for cancer therapy. Finally, it introduced a new perception of EMT biological meaning based on an evolutionary perspective. The meeting integrated the TEMTIA general assembly , allowing general discussion about the future of the association, starting with the site of the next meeting, now decided to take place in Seattle (US), late 2024.

Mutual regulation of TGFß-induced oncogenic EMT, cell cycle progression and the DDR.

TGFß signaling and the DNA damage response (DDR) are two cellular toolboxes with a strong impact on cancer biology. While TGFß as a pleiotropic cytokine affects essentially all hallmarks of cancer, the multifunctional DDR mostly orchestrates cell cycle progression, DNA repair, chromatin remodeling and cell death. One oncogenic effect of TGFß is the partial activation of epithelial-to-mesenchymal transition (EMT), conferring invasiveness, cellular plasticity and resistance to various noxae. Several reports show that both individual networks as well as their interface affect chemo-/radiotherapies. However, the underlying mechanisms remain poorly resolved. EMT often correlates with TGFß-induced slowing of proliferation, yet numerous studies demonstrate that particularly the co-activated EMT transcription factors counteract anti-proliferative signaling in a partially non-redundant manner. Collectively, evidence piled up over decades underscore a multifaceted, reciprocal inter-connection of TGFß signaling / EMT with the DDR / cell cycle progression, which we will discuss here. Altogether, we conclude that full cell cycle arrest is barely compatible with the propagation of oncogenic EMT traits and further propose that 'EMT-linked DDR plasticity' is a crucial, yet intricate facet of malignancy, decisively affecting metastasis formation and therapy resistance.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/ß-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Multi-cancer analysis reveals universal association of oncogenic LBH expression with DNA hypomethylation and WNT-Integrin signaling pathways.

Limb-Bud and Heart (LBH) is a developmental transcription co-factor deregulated in cancer, with reported oncogenic and tumor suppressive effects. However, LBH expression in most cancer types remains unknown, impeding understanding of its mechanistic function Here, we performed systematic bioinformatic and TMA analysis for LBH in >20 different cancer types. LBH was overexpressed in most cancers compared to normal tissues (>1.5-fold; p < 0.05), including colon-rectal, pancreatic, esophageal, liver, stomach, bladder, kidney, prostate, testicular, brain, head & neck cancers, and sarcoma, correlating with poor prognosis. The cancer types showing LBH downregulation were lung, melanoma, ovarian, cervical, and uterine cancer, while both LBH over- and under-expression were observed in hematopoietic malignancies. In cancers with LBH overexpression, the LBH locus was frequently hypomethylated, identifying DNA hypomethylation as a potential mechanism for LBH dysregulation. Pathway analysis identified a universal, prognostically significant correlation between LBH overexpression and the WNT-Integrin signaling pathways. Validation of the clinical association of LBH with WNT activation in gastrointestinal cancer cell lines, and in colorectal patient samples by IHC uncovered that LBH is specifically expressed in tumor cells with nuclear beta-catenin at the invasive front. Collectively, these data reveal a high degree of LBH dysregulation in cancer and establish LBH as pan-cancer biomarker for detecting WNT hyperactivation in clinical specimens.

A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

The EMT transcription factor ZEB1 governs a fitness-promoting but vulnerable DNA replication stress response.

The DNA damage response (DDR) and epithelial-to-mesenchymal transition (EMT) are two crucial cellular programs in cancer biology. While the DDR orchestrates cell-cycle progression, DNA repair, and cell death, EMT promotes invasiveness, cellular plasticity, and intratumor heterogeneity. Therapeutic targeting of EMT transcription factors, such as ZEB1, remains challenging, but tumor-promoting DDR alterations elicit specific vulnerabilities. Using multi-omics, inhibitors, and high-content microscopy, we discover a chemoresistant ZEB1-high-expressing sub-population (ZEB1hi) with co-rewired cell-cycle progression and proficient DDR across tumor entities. ZEB1 stimulates accelerated S-phase entry via CDK6, inflicting endogenous DNA replication stress. However, DDR buildups involving constitutive MRE11-dependent fork resection allow homeostatic cycling and enrichment of ZEB1hi cells during transforming growth factor ß (TGF-ß)-induced EMT and chemotherapy. Thus, ZEB1 promotes G1/S transition to launch a progressive DDR benefitting stress tolerance, which concurrently manifests a targetable vulnerability in chemoresistant ZEB1hi cells. Our study thus highlights the translationally relevant intercept of the DDR and EMT.

The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of ß-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1.

The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1-ezrin-NF-κB signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-κB signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Deregulation of Transcription Factor Networks Driving Cell Plasticity and Metastasis in Pancreatic Cancer.

Pancreatic cancer is a very aggressive disease with 5-year survival rates of less than 10%. The constantly increasing incidence and stagnant patient outcomes despite changes in treatment regimens emphasize the requirement of a better understanding of the disease mechanisms. Challenges in treating pancreatic cancer include diagnosis at already progressed disease states due to the lack of early detection methods, rapid acquisition of therapy resistance, and high metastatic competence. Pancreatic ductal adenocarcinoma, the most prevalent type of pancreatic cancer, frequently shows dominant-active mutations in KRAS and TP53 as well as inactivation of genes involved in differentiation and cell-cycle regulation (e.g. SMAD4 and CDKN2A). Besides somatic mutations, deregulated transcription factor activities strongly contribute to disease progression. Specifically, transcriptional regulatory networks essential for proper lineage specification and differentiation during pancreas development are reactivated or become deregulated in the context of cancer and exacerbate progression towards an aggressive phenotype. This review summarizes the recent literature on transcription factor networks and epigenetic gene regulation that play a crucial role during tumorigenesis.

Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

Dynamic EMT: a multi-tool for tumor progression.

The process of epithelial-mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non-classical functions, and introduce EMT as a true tumorigenic multi-tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will-if acknowledging these complexities-be a possibility to concurrently interfere with tumor progression on many levels.

The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus.

Radial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division.

Pancreas morphogenesis and homeostasis depends on tightly regulated Zeb1 levels in epithelial cells.

The pancreas is comprised of exocrine and endocrine compartments releasing digestive enzymes into the duodenum and regulating blood glucose levels by insulin and glucagon release. Tissue homeostasis is depending on transcription factor networks, involving Ptf1α, Ngn3, Nkx6.1, and Sox9, which are already activated during organogenesis. However, proper organ function is challenged by diets of high sugar and fat content, increasing the risk of type 2 diabetes and other disorders. A detailed understanding of processes that are important for homeostasis and are impaired during type 2 diabetes is lacking. Here, we show that Zeb1-a transcription factor known for its pivotal role in epithelial-mesenchymal transition, cell plasticity, and metastasis in cancer-is expressed at low levels in epithelial cells of the pancreas and is crucial for organogenesis and pancreas function. Loss of Zeb1 in these cells result in an increase of islet mass, impaired glucose tolerance, and sensitizes to develop liver and pancreas steatosis during diabetes and obesity. Interestingly, moderate overexpression of Zeb1 results in severe pancreas agenesis and lethality after birth, due to islet insufficiency and lack of acinar structures. We show that Zeb1 induction interferes with proper differentiation, cell survival, and proliferation during pancreas formation, due to deregulated expression of endocrine-specific transcription factors. In summary, our analysis suggests a novel role of Zeb1 for homeostasis in epithelial cells that is indispensable for pancreas morphogenesis and proper organ function involving a tight regulation of Zeb1 expression.

Cytomegalovirus subverts macrophage identity.

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.