Cone Beam Computed Tomography of mandibular bone density in non-diabetic vs. diabetic populations.

BACKGROUND: The successful placement of dental implant largely depends on the properties of alveolar bone at the recipient site. Systemic conditions such as diabetes mellitus could impair bone quality and compromise implant treatment. However, limited information in this area exists so far. The objective of the study is to use cone beam computed tomography (CBCT) to assess mandibular bone mineral density (BMD) in diabetic and non-diabetic populations. METHODS: The patients who had CBCT scans in the school from 2011-2015 were screened, and 14 diabetic and 14 non- diabetic patients with matched age, gender, and ethnicity were recruited. BMD was measured on reconstructed CBCT sagittal views at 7 mm2 rectangular areas on 6 sites for each patient. For cortical bone, BMD was measured at the inferior border of mandible in the midline and between the first and second premolar bilaterally. For cancellous bone, BMD was measured in the midline of mandible halfway between the lingual foramen/canal and the inferior border of mandible, and at the premolar area halfway between the mandibular canal and the inferior border of mandible bilaterally. For diabetic patients, the glycosylated hemoglobin (HbAlc) values were obtained after informed consent. Statistical significant difference was set at p <0.05. The correlation between BMD and the age, gender, and HbAlc value of the patients was analyzed. An institutional IRB approval was obtained for the study. RESULTS: Diabetic patients had significantly lower cancellous BMD than non-diabetic subjects in the posterior mandibles (367 vs. 430, p<0.05). For both groups, cancellous BMD in the posterior mandible was significantly lower than that of anterior mandible. CONCLUSIONS: Diabetic patients have decreased BMD in the posterior mandible which could adversely affect implant placement at these areas.

Immunobiology and immunotherapeutic targeting of glioma stem cells.

For decades human brain tumors have confounded our efforts to effectively manage and treat patients. In adults, glioblastoma multiforme is the most common malignant brain tumor with a patient survival of just over 14 months. In children, brain tumors are the leading cause of solid tumor cancer death and gliomas account for one-fifth of all childhood cancers. Despite advances in conventional treatments such as surgical resection, radiotherapy, and systemic chemotherapy, the incidence and mortality rates for gliomas have essentially stayed the same. Furthermore, research efforts into novel therapeutics that initially appeared promising have yet to show a marked benefit. A shocking and somewhat disturbing view is that investigators and clinicians may have been targeting the wrong cells, resulting in the appearance of the removal or eradication of patient gliomas only to have brain cancer recurrence. Here we review research progress in immunotherapy as it pertains to glioma treatment and how it can and is being adapted to target glioma stem cells (GSCs) as a means of dealing with this potential paradigm.

Applications and current challenges of chimeric antigen receptor T cells in treating high-grade gliomas in adult and pediatric populations.

High-grade gliomas (HGGs) continue to be some of the most devastating diseases in the USA. Despite extensive efforts, the survival of HGG patients has remained relatively stagnant. Chimeric antigen receptor (CAR) T-cell immunotherapy has recently been studied in the context of improving these tumors' clinical outcomes. HGG murine models treated with CAR T cells targeting tumor antigens have shown reduced tumor burden and longer overall survival than models without treatment. Subsequent clinical trials investigating the efficacy of CAR T cells have further shown that this therapy could be safe and might reduce tumor burden. However, there are still many challenges that need to be addressed to optimize the safety and efficacy of CAR T-cell therapy in treating HGG patients.

This publication describes the current application of chimeric antigen T-cell (CAR T-cell) therapy in treating high-grade gliomas (HGGs). Treatment of various HGG models with CAR T cells has shown that this therapy is often able to shrink HGG tumors and prolong the survival of these models. Subsequent clinical trials have shown that CAR T-cell therapy can reduce tumor size in some HGG patients. Patients in these clinical trials have tolerated the treatment well, though more robust studies are needed to confirm this treatment's safety. Additionally, other challenges, such as getting CAR T cells into the brain and to the tumor, need to be addressed to improve the effectiveness of this therapy for HGG patients.

Characterization of neural mechanotransduction response in human traumatic brain injury organoid model.

The ability to model physiological systems through 3D neural in-vitro systems may enable new treatments for various diseases while lowering the need for challenging animal and human testing. Creating such an environment, and even more impactful, one that mimics human brain tissue under mechanical stimulation, would be extremely useful to study a range of human-specific biological processes and conditions related to brain trauma. One approach is to use human cerebral organoids (hCOs) in-vitro models. hCOs recreate key cytoarchitectural features of the human brain, distinguishing themselves from more traditional 2D cultures and organ-on-a-chip models, as well as in-vivo animal models. Here, we propose a novel approach to emulate mild and moderate traumatic brain injury (TBI) using hCOs that undergo strain rates indicative of TBI. We subjected the hCOs to mild (2 s[Formula: see text]) and moderate (14 s[Formula: see text]) loading conditions, examined the mechanotransduction response, and investigated downstream genomic effects and regulatory pathways. The revealed pathways of note were cell death and metabolic and biosynthetic pathways implicating genes such as CARD9, ENO1, and FOXP3, respectively. Additionally, we show a steeper ascent in calcium signaling as we imposed higher loading conditions on the organoids. The elucidation of neural response to mechanical stimulation in reliable human cerebral organoid models gives insights into a better understanding of TBI in humans.

Human brain organoid code of conduct.

Human brain organoids are models derived from human embryonic or induced pluripotent stem cells that mimic basic cerebral microanatomy and demonstrate simple functional neuronal networks. Brain organoids have been a rapidly expanding avenue for biomedical research in general and specifically: neural development, regeneration, and central nervous system pathophysiology. However, technology replicating functional aspects of the human brain, including electrically active neural networks, requires a responsible code of conduct. In this review, we focus the discussion on intrinsic and extrinsic ethical factors associated with organoids: intrinsic considerations arise with the growing complexity of human brain organoids, including human-animal chimerism, consciousness development, and questions of where these human-like beings fall in a moral hierarchy. Extrinsic considerations explore ethics on obtainment, manufacturing, and production of sophisticated human products. In summary, a thoughtful code of conduct using human brain organoids towards the advancement of science and medicine is crucial. This article shall facilitate a structured thought process approaching the moral landscape of organoid technology.

TP53-PTEN-NF1 depletion in human brain organoids produces a glioma phenotype in vitro.

Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.

ZEB1 loss increases glioma stem cell tumorigenicity and resistance to chemoradiation.

OBJECTIVE: Glioblastoma has been known to be resistant to chemotherapy and radiation, whereas the underlying mechanisms of resistance have not been fully elucidated. The authors studied the role of the transcription factor ZEB1 (zinc finger E-box-binding homeobox 1 protein), which is associated with epithelial-mesenchymal transition (EMT) and is central to the stemness of glioblastoma, to determine its role in therapeutic resistance to radiation and chemotherapy. The authors previously demonstrated that ZEB1 is deleted in a majority of glioblastomas. METHODS: The authors explored resistance to therapy in the context of ZEB1 loss and overexpression in glioma stem cells (GSCs) and in patient data. RESULTS: Patients with ZEB1 loss had a shorter survival time than patients with wild-type ZEB1 in both the high- and low-MGMT groups. Consistent with the clinical data, mice implanted with ZEB1 knockdown GSCs showed shortened survival compared with mice inoculated with nonsilencing control (NS) short-hairpin RNA (shRNA) GSC glioblastoma. ZEB1-deleted GSCs demonstrated increased tumorigenicity with regard to proliferation and invasion. Importantly, GSCs that lose ZEB1 expression develop enhanced resistance to chemotherapy, radiotherapy, and combined chemoradiation. ZEB1 loss may lead to increased HER3 expression through the HER3/Akt pathway associated with this chemoresistance. Conversely, overexpression of ZEB1 in GSCs that are ZEB1 null leads to increased sensitivity to chemoradiation. CONCLUSIONS: The study results indicate that ZEB1 loss in cancer stem cells confers resistance to chemoradiation and uncovers a potentially targetable cell surface receptor in these resistant cells.

The Subventricular Zone in Glioblastoma: Genesis, Maintenance, and Modeling.

Glioblastoma (GBM) is a malignant tumor with a median survival rate of 15-16 months with standard care; however, cases of successful treatment offer hope that an enhanced understanding of the pathology will improve the prognosis. The cell of origin in GBM remains controversial. Recent evidence has implicated stem cells as cells of origin in many cancers. Neural stem/precursor cells (NSCs) are being evaluated as potential initiators of GBM tumorigenesis. The NSCs in the subventricular zone (SVZ) have demonstrated similar molecular profiles and share several distinctive characteristics to proliferative glioblastoma stem cells (GSCs) in GBM. Genomic and proteomic studies comparing the SVZ and GBM support the hypothesis that the tumor cells and SVZ cells are related. Animal models corroborate this connection, demonstrating migratory patterns from the SVZ to the tumor. Along with laboratory and animal research, clinical studies have demonstrated improved progression-free survival in patients with GBM after radiation to the ipsilateral SVZ. Additionally, key genetic mutations in GBM for the most part carry regulatory roles in the SVZ as well. An exciting avenue towards SVZ modeling and determining its role in gliomagenesis in the human context is human brain organoids. Here we comprehensively discuss and review the role of the SVZ in GBM genesis, maintenance, and modeling.

Navigated 3D ultrasound-guided resection of high-grade gliomas: A case series and review.

Background: The crux in high-grade glioma surgery remains maximizing resection without affecting eloquent brain areas. Toward this, a myriad of adjunct tools and techniques has been employed to enhance surgical safety and efficacy. Despite intraoperative MRI and advanced neuronavigational techniques, as well as augmented reality, to date, the only true real-time visualization tool remains the ultrasound (US). Neuroultrasonography is a cost-efficient imaging modality that offers instant, real-time information about the changing anatomical landscape intraoperatively. Recent advances in technology now allow for the integration of intraoperative US with neuronavigation. Case Description: In this report, we present the resection technique for three cases of high-grade gliomas (two glioblastomas and one anaplastic astrocytoma). The patient presented with a variable clinical spectrum. All three cases have been performed using the Brainlab® neuronavigation system (BrainLAB, Munich, Germany) and the bk5000 US Machine® (BK Medical, Analogic Corporation, Peabody, Massachusetts, USA). Conclusion: Gross total resection was achieved in all three cases. The use of 3D navigated US was a reliable adjunct surgical tool in achieving favorable resection outcomes in these patients.

The Evolving Role of Induced Pluripotent Stem Cells and Cerebral Organoids in Treating and Modeling Neurosurgical Diseases.

Over the past decade, the use of induced pluripotent stem cells (IPSCs), as both direct therapeutics and building blocks for 3D in vitro models, has exhibited exciting potential in both helping to elucidate pathogenic mechanisms and treating diseases relevant to neurosurgery. Transplantation of IPSCs is being studied in neurological injuries and diseases, such as spinal cord injury and Parkinson's disease, whose clinical manifestations stem from underlying neuronal and/or axonal degeneration. Both animal models and clinical trials have shown that IPSCs have the ability to regenerate damaged neural tissue. Such evidence makes IPSCs a potentially promising therapeutic modality for patients who suffer from these neurological injuries/diseases. In addition, the cerebral organoid, a 3D assembly of IPSC aggregates that develops heterogeneous brain regions, has become the first in vitro model to closely recapitulate the complexity of the brain extracellular matrix, a 3-dimensional network of molecules that structurally and biochemically support neighboring cells. Cerebral organoids have become an exciting prospect for modeling and testing drug susceptibility of brain tumors, such as glioblastoma and metastatic brain cancer. As patient-derived organoid models are becoming more faithful to the brain, they are becoming an increasingly accurate substitute for patient clinical trials; such patient-less trials would protect the patient from potentially ineffective drugs, and speed up trial results and optimize cost. In this review, we aim to describe the role of IPSCs and cerebral organoids in treating and modeling diseases that are relevant to neurosurgery.

CD133 mRNA-transfected dendritic cells induce coordinated cytotoxic and helper T cell responses against breast cancer stem cells.

Breast cancer, a leading cause of death yearly, has been shown to be initiated and propagated by cancer stem cells. CD133, a cell surface antigen, has been shown to be present on cancer stem cells of many solid tumors, including breast cancer. A limitation to targeting CD133 is major histocompatibility complex (MHC)-restricted presentation of epitopes, leading to activation of only one arm of the immune system: either CD4+ helper T cells or CD8+ cytotoxic T cells. Thus, we hypothesized that by creating an MHC-independent vaccination, we would give rise to a sustained immune response against CD133 in triple-negative breast cancer (TNBCs). We transfected CD133 mRNA into dendritic cells and then tested this in animal models of TNBC. We showed in these models the activation of both CD8+ cytotoxic T cells and CD4+ helper T cells by dendritic cell vaccination with modified CD133 mRNA, with subsequent decrease in tumor growth. This study for the first time demonstrates in a syngeneic mouse model of TNBC that targeting CD133, in an MHC-independent manner, is an effective strategy against the cancer stem cell population, leading to tumor abrogation.

Elevated Asparagine Biosynthesis Drives Brain Tumor Stem Cell Metabolic Plasticity and Resistance to Oxidative Stress.

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

CD133 mRNA-Loaded Dendritic Cell Vaccination Abrogates Glioma Stem Cell Propagation in Humanized Glioblastoma Mouse Model.

Cancer stem cells are initiating cells of cancer and propagate its growth through self-renewal and differentiation of its daughter cells. CD133 is a cell surface antigen that is present on glioma stem cells and has been used to prospectively isolate glioma stem cells. We hypothesized that a major histocompatibility complex (MHC)-independent and long-lasting immune response against CD133 could be generated by transfecting CD133 mRNA into dendritic cells and vaccinating animals with experimental gliomas. To test this hypothesis, we developed a novel humanized mouse model using CD34-positive hematopoietic stem cells. We confirmed the robust simultaneous activation of CD8- and CD4-positive T cells by dendritic cell vaccination with modified CD133 mRNA leading to a potent and long-lived immune response, with subsequent abrogation of CD133-positive glioma stem cell propagation and tumor growth. This study for the first time demonstrates in both a humanized mouse model and in a syngeneic mouse model of glioblastoma that targeting a glioma stem cell-associated antigen is an effective strategy to target and kill glioma stem cells. This novel and simple humanized mouse model for immunotherapy is a significant advance in our ability to test human-specific immunotherapies for glioblastoma.

Tumor Microenvironment Is Critical for the Maintenance of Cellular States Found in Primary Glioblastomas.

Glioblastoma (GBM), an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA sequencing of tumor cells from 5 patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models. SIGNIFICANCE: It has been unclear how well different patient-derived GBM models are able to recreate the full heterogeneity of primary tumors. Here, we provide a complete transcriptomic characterization of the major model types. We show that the microenvironment is crucial for recapitulating GSC cellular states, highlighting the importance of tumor-host cell interactions.See related commentary by Luo and Weiss, p. 907.This article is highlighted in the In This Issue feature, p. 890.

QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model.

INTRODUCTION: Substantial preclinical evidence has indicated that inhibition of integrin linked-kinase (ILK) correlates with cytotoxic/cytostatic cellular effects, delayed tumor growth in animal models of cancer, and inhibition of angiogenesis. Widely anticipated to represent a very promising therapeutic target in several cancer indications, it is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. The purpose of this study was to investigate the therapeutic potential of the ILK small molecule inhibitor, QLT0267 (267), alone or in combination with chemotherapies commonly used to treat breast cancer patients. METHODS: A single end-point metabolic assay was used as an initial screen for 267 interactions with selected chemotherapeutic agents. These in vitro assays were completed with seven breast cancer cell lines including several which over-expressed human epidermal growth factor receptor 2 (Her2). One agent, docetaxel (Dt), consistently produced synergistic interactions when combined with 267. Dt/267 interactions were further characterized by measuring therapeutic endpoints linked to phosphorylated protein kinase B (P-AKT) suppression, inhibition of vascular endothelial growth factor (VEGF) secretion and changes in cytoarchitecture. In vivo efficacy studies were completed in mice bearing orthotopic xenografts where tumor growth was assessed by bioluminescence and calliper methods. RESULTS: The combination of 267 and Dt resulted in increased cytotoxic activity, as determined using an assay of metabolic activity. Combinations of cisplatin, doxorubicin, vinorelbine, paclitaxel, and trastuzumab produced antagonistic interactions. Further endpoint analysis in cell lines with low Her2 levels revealed that the 267/Dt combinations resulted in: a three-fold decrease in concentration (dose) of 267 required to achieve 50% inhibition of P-AKT; and a dramatic disruption of normal filamentous-actin cellular architecture. In contrast to Her2-positive cell lines, three-fold higher concentrations of 267 were required to achieve 50% inhibition of P-AKT when the drug was used in combination with Dt. In vivo studies focusing on low Her2-expressing breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with 267/Dt engendered improved therapeutic effects when compared with mice treated with either agent alone. CONCLUSIONS: The findings indicate that the 267/Dt drug combination confers increased (synergistic) therapeutic efficacy towards human breast cancer cells that express low levels of Her2.

Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK).

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1alpha). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser473), secreted VEGF, G2-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm3 versus control 549 mm3). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1alpha expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas.

Modeling Patient-Derived Glioblastoma with Cerebral Organoids.

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs.

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-kappaB, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex.

The Curious Case of ZEB1.

The Zinc Finger E-box binding homeobox (ZEB1/TCF8 or DeltaEF1) is at the forefront of transcription factors involved in controlling epithelial-to-mesenchymal transitions (EMT). Essentially, EMT allows for the reorganization of epithelial cells to become migratory cells with a mesenchymal phenotype.  In addition to ZEB1 being involved in embryonic development, ZEB1 has also been linked to processes involving micro-RNAs, long non-coding RNAs and stem cells. In recent years there has been an accumulation of evidence with regard to ZEB1 in various cancers. Although increased ZEB1 expression has largely been associated with EMT, cancer invasion, and tumorigenicity, there have been some episodic reports that have gone against the traditional reporting of the role of ZEB1. Indicating that the function of ZEB1 and the mechanisms by which ZEB1 facilitates its activities is more complex than was once appreciated. This complexity is further exacerbated by the notion that ZEB1 can act not only as a transcriptional repressor but a transcriptional activator as well. This review seeks to shed light on the complexity of ZEB1 with respect to cancer.