The FOXC2 Transcription Factor: A Master Regulator of Chemoresistance in Cancer.

FOXC2, a member of the forkhead box family of transcription factors, is an emerging oncogene that has been linked to several hallmarks of cancer progression. Among its many oncogenic functions is the promotion of drug resistance, with evidence supporting roles for FOXC2 in escape from broad classes of chemotherapeutics across an array of cancer types. In this Mini-Review, we highlight the current understanding of the mechanisms by which FOXC2 drives cancer chemoresistance, including its roles in the promotion of epithelial-mesenchymal transition, induction of multidrug transporters, activation of the oxidative stress response, and deregulation of cell survival signaling pathways. We discuss the clinical implications of these findings, including strategies for modulating FOXC2-associated chemoresistance in cancer. Particular attention is given to ways in which FOXC2 and its downstream gene products and pathways can be targeted to restore chemosensitivity in cancer cells. In addition, the utility of FOXC2 expression as a predictor of patient response to chemotherapy is also highlighted, with emphasis on the value of FOXC2 as a novel biomarker that can be used to guide therapeutic choice towards regimens most likely to achieve clinical benefit during frontline therapy.

Genetic dysregulation of immunologic and oncogenic signaling pathways associated with tumor-intrinsic immune resistance: a molecular basis for combination targeted therapy-immunotherapy for cancer.

Since the turn of the century, advances in targeted therapy and immunotherapy have revolutionized the treatment of cancer. Although these approaches have far outperformed traditional therapies in various clinical settings, both remain plagued by mechanisms of innate and acquired resistance that limit therapeutic efficacy in many patients. With a focus on tumor-intrinsic resistance to immunotherapy, this review highlights our current understanding of the immunologic and oncogenic pathways whose genetic dysregulation in cancer cells enables immune escape. Emphasis is placed on genomic, epigenomic, transcriptomic, and proteomic aberrations that influence the activity of these pathways in the context of immune resistance. Specifically, the role of pathways that govern interferon signaling, antigen processing and presentation, and immunologic cell death as determinants of tumor immune susceptibility are discussed. Likewise, mechanisms of tumor immune resistance mediated by dysregulated RAS-MAPK, WNT, PI3K-AKT-mTOR, and cell cycle pathways are described. Finally, this review highlights the ways in which recent insight into genetic dysregulation of these immunologic and oncogenic signaling pathways is informing the design of combination targeted therapy-immunotherapy regimens that aim to restore immune susceptibility of cancer cells by overcoming resistance mechanisms that often limit the success of monotherapies.

A Bioinformatic Approach to Enhance Undergraduate Student Understanding of the Cancer-Immunity Cycle.

Recent advances in tumor immunology and cancer immunotherapy have generated significant interest in the field of immuno-oncology. With the promise of these advances comes an increasing need to train the next generation of scientists who will support ongoing basic and clinical research efforts in this field. At this time, however, there remains a documented underrepresentation of tumor immunology as a core content area in many undergraduate science curricula. This study introduces a novel pedagogical strategy that aimed to promote undergraduate student interest in tumor immunology in ways that support recent education guidelines published by the American Association of Immunologists, and it highlights the efficacy of this approach in enhancing student understanding of concepts relevant to the Cancer-Immunity Cycle. Using RNA-sequencing data obtained from clinical specimens catalogued in The Cancer Genome Atlas, students performed Kaplan-Meier survival analyses to identify Cancer-Immunity Cycle genes with prognostic significance. After correlating expression of such genes with tumor-infiltrating immune cell populations using a bioinformatic tool to deconvolute whole tumor-transcriptome data, students undertook an exercise that requires integration of course content and findings from the primary literature to generate hypotheses about the influence of genetic factors and immune cell types on the Cancer-Immunity Cycle and overall patient outcome. A pre-/post-project assessment instrument demonstrated the efficacy of this approach as a means of improving undergraduate student understanding of core cancer immunology concepts. This report describes these data and discusses potential ways in which the project can be adapted to extend its utility to broad and diverse student populations.

Oncogenic functions of the FOXC2 transcription factor: a hallmarks of cancer perspective.

Epigenetic regulation of gene expression is a fundamental determinant of molecular and cellular function, and epigenetic reprogramming in the context of cancer has emerged as one of the key enabling characteristics associated with acquisition of the core hallmarks of this disease. As such, there has been renewed interest in studying the role of transcription factors as epigenetic regulators of gene expression in cancer. In this review, we discuss the current state of knowledge surrounding the oncogenic functions of FOXC2, a transcription factor that frequently becomes dysregulated in a variety of cancer types. In addition to highlighting the clinical impact of aberrant FOXC2 activity in cancer, we discuss mechanisms by which this transcription factor becomes dysregulated in both tumor and tumor-associated cells, placing particular emphasis on the ways in which FOXC2 promotes key hallmarks of cancer progression. Finally, we bring attention to important issues related to the oncogenic dysregulation of FOXC2 that must be addressed going forward in order to improve our understanding of FOXC2-mediated cancer progression and to guide prognostic and therapeutic applications of this knowledge in clinical settings.

Using The Cancer Genome Atlas as a Tool to Improve Undergraduate Student Understanding of Cancer Genetics and the Hallmarks of Cancer Progression.

Undergraduate students harbor a number of misconceptions about the complexity of cancer. Though educational interventions have been shown to promote student learning of various aspects of this ubiquitous disease, to date no studies have evaluated undergraduate student understanding of the Hallmarks of Cancer, a set of core properties that have emerged as the defining characteristics that drive cancer development and progression. This study documents poor baseline knowledge of many fundamental aspects of cancer biology and genetics by 2nd- and 3rd-year undergraduate students, and it evaluates the efficacy of a student-centered project as a pedagogical strategy for improving student understanding of both the hallmarks of cancer and the genetic influences that drive their acquisition by cancer cells. Using genomic and transcriptomic datasets from The Cancer Genome Atlas, students investigated the most common genetic aberrations associated with specific cancer types of interest and then researched the functions of these altered genes in order to link their aberrations with specific hallmark properties of cancer. A pre-/post-project assessment of student understanding of topics related to the hallmarks of cancer demonstrated the efficacy of this approach as a means of educating undergraduate students about core cancer concepts.

A call for discovery: Re-envisioning The Cancer Genome Atlas as a blueprint for a TCGA2.0-The COVID-19 Genome Atlas.

The COVID-19 pandemic has impacted the health of millions and had a myriad of devastating consequences for global societies since its emergence in 2019. Noting parallels between the impact of COVID-19 and cancer as diseases of global health significance, and as a way of building off the successes of The Cancer Genome Atlas (TCGA) as a comprehensive, multiomics approach to understand and combat cancer, this Call For Discovery provides a vision for creating a new TCGA2.0 (The COVID-19 Genome Atlas) as a tool that will benefit researchers, clinicians, and patients alike as the scientific community works to better understand not only the various determinants of COVID-19 disease outcome but also the most effective ways to manage and treat COVID-19 disease complications.

Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing.

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.

A Flow Cytometric Assay for Investigating Melanoma Cell Adhesion to Lymphatic Endothelial Cells.

Lymph node invasion by tumor cells is an important process in the progression of melanoma and is a poor prognostic factor for patients with this cancer. Before they are able to spread to regional lymph nodes, though, melanoma cells must first adhere to lymphatic endothelium and transmigrate into the lymphatic vasculature. In order to study melanoma cell adhesion to lymphatic endothelial cells and the factors that regulate this process, we have developed an in vitro flow cytometry-based assay to measure melanoma cell attachment to lymphatic endothelial cells. This assay will be a useful tool for investigating the interactions that take place between melanoma cells and lymphatic endothelial cells during the adhesion process.

The prognostic significance of FOXC2 gene expression in cancer: A comprehensive analysis of RNA-seq data from the cancer genome atlas.

The FOXC2 transcription factor is a key regulator of tumor progression in many cancer types. Known to exhibit an array of oncogenic functions when dysregulated, FOXC2 has emerged as a useful biomarker for predicting disease aggression and patient outcome. In this regard, increased expression and nuclear localization of FOXC2 protein in tumor tissue have become well-established as poor prognostic factors for many cancer types. However, whether FOXC2 gene expression can serve as a similarly useful RNA-level biomarker has remained largely unexplored. Therefore, we conducted a comprehensive analysis of TCGA RNA-seq data to evaluate whether FOXC2 gene expression levels in primary tumor biopsies correlate with patient outcome. We report herein that increased expression of FOXC2 RNA in tumor tissue is a poor prognostic factor for patient survival in many cancer types. Moreover, we also found that FOXC2 gene expression predicts cancer patient response to several commonly prescribed chemotherapeutics. Together, these data highlight FOXC2 RNA expression in tumor tissue as an important biomarker with prognostic significance for solid tumors of diverse origin.

Genomic and transcriptional changes in IFNγ pathway genes are putative biomarkers of response to ipilimumab immunotherapy in melanoma patients.

Evaluation of: Gao J, Shi LZ, Zhao H et al. Loss of IFN-γ pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy. Cell 167(2), 397-404 (2016). Tumor resistance to immune checkpoint inhibitors limits therapeutic efficacy in many cancer patients. The study by Gao et al. highlighted herein describes a fundamental mechanism by which melanoma escapes the CTLA-4-targeting drug ipilimumab, the first FDA-approved immune checkpoint inhibitor for cancer. This work describes genomic alterations to IFNγ signaling pathway components as a mechanism of melanoma resistance to ipilimumab and has spawned several studies documenting the significance of this pathway to the efficacy of immunotherapy. The authors highlight new analysis of TCGA RNA-seq data that both support and extend the relevance of the IFNγ pathway to CTLA-4 checkpoint blockade as first introduced by this seminal paper, and the authors discuss the relevance of these collective findings to the future of cancer immunotherapy.

Tumor microenvironmental influences on dendritic cell and T cell function: A focus on clinically relevant immunologic and metabolic checkpoints.

Cancer immunotherapy is fast becoming one of the most promising means of treating malignant disease. Cancer vaccines, adoptive cell transfer therapies, and immune checkpoint blockade have all shown varying levels of success in the clinical management of several cancer types in recent years. However, despite the clinical benefits often achieved by these regimens, an ongoing problem for many patients is the inherent or acquired resistance of their cancer to immunotherapy. It is now appreciated that dendritic cells and T lymphocytes both play key roles in antitumor immune responses and that the tumor microenvironment presents a number of barriers to the function of these cells that can ultimately limit the success of immunotherapy. In particular, the engagement of several immunologic and metabolic checkpoints within the hostile tumor microenvironment can severely compromise the antitumor functions of these important immune populations. This review highlights work from both preclinical and clinical studies that has shaped our understanding of the tumor microenvironment and its influence on dendritic cell and T cell function. It focuses on clinically relevant targeted and immunotherapeutic strategies that have emerged from these studies in an effort to prevent or overcome immune subversion within the tumor microenvironment. Emphasis is also placed on the potential of next-generation combinatorial regimens that target metabolic and immunologic impediments to dendritic cell and T lymphocyte function as strategies to improve antitumor immune reactivity and the clinical outcome of cancer immunotherapy going forward.

The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness.

BACKGROUND/AIM: The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. MATERIALS AND METHODS: We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. RESULTS: FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. CONCLUSION: FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies.

Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors.

Although T lymphocytes have long been appreciated for their role in the immunosurveillance of cancer, it has been the realization that cancer cells may ultimately escape a response from tumor-reactive T cells that has ignited efforts to enhance the efficacy of anti-tumor immune responses. Recent advances in our understanding of T cell immunobiology have been particularly instrumental in informing therapeutic strategies to overcome mechanisms of tumor immune escape, and immune checkpoint blockade has emerged as one of the most promising therapeutic options for patients in the history of cancer treatment. Designed to interfere with inhibitory pathways that naturally constrain T cell reactivity, immune checkpoint blockade releases inherent limits on the activation and maintenance of T cell effector function. In the context of cancer, where negative T cell regulatory pathways are often overactive, immune checkpoint blockade has proven to be an effective strategy for enhancing the effector activity and clinical impact of anti-tumor T cells. Checkpoint inhibitors targeting CTLA-4, PD-1, and PD-L1 have yielded unprecedented and durable responses in a significant percentage of cancer patients in recent years, leading to U.S. FDA approval of six checkpoint inhibitors for numerous cancer indications since 2011. In this review, we highlight the clinical success of these FDA-approved immune checkpoint inhibitors and discuss current challenges and future strategies that must be considered going forward to maximize the efficacy of immune checkpoint blockade therapy for cancer.

Strategies to Improve the Efficacy of Dendritic Cell-Based Immunotherapy for Melanoma.

Melanoma is a highly aggressive form of skin cancer that frequently metastasizes to vital organs, where it is often difficult to treat with traditional therapies such as surgery and radiation. In such cases of metastatic disease, immunotherapy has emerged in recent years as an exciting treatment option for melanoma patients. Despite unprecedented successes with immune therapy in the clinic, many patients still experience disease relapse, and others fail to respond at all, thus highlighting the need to better understand factors that influence the efficacy of antitumor immune responses. At the heart of antitumor immunity are dendritic cells (DCs), an innate population of cells that function as critical regulators of immune tolerance and activation. As such, DCs have the potential to serve as important targets and delivery agents of cancer immunotherapies. Even immunotherapies that do not directly target or employ DCs, such as checkpoint blockade therapy and adoptive cell transfer therapy, are likely to rely on DCs that shape the quality of therapy-associated antitumor immunity. Therefore, understanding factors that regulate the function of tumor-associated DCs is critical for optimizing both current and future immunotherapeutic strategies for treating melanoma. To this end, this review focuses on advances in our understanding of DC function in the context of melanoma, with particular emphasis on (1) the role of immunogenic cell death in eliciting tumor-associated DC activation, (2) immunosuppression of DC function by melanoma-associated factors in the tumor microenvironment, (3) metabolic constraints on the activation of tumor-associated DCs, and (4) the role of the microbiome in shaping the immunogenicity of DCs and the overall quality of anti-melanoma immune responses they mediate. Additionally, this review highlights novel DC-based immunotherapies for melanoma that are emerging from recent progress in each of these areas of investigation, and it discusses current issues and questions that will need to be addressed in future studies aimed at optimizing the function of melanoma-associated DCs and the antitumor immune responses they direct against this cancer.

Dysregulation of TGFß1 Activity in Cancer and Its Influence on the Quality of Anti-Tumor Immunity.

TGFß1 is a pleiotropic cytokine that exhibits a variety of physiologic and immune regulatory functions. Although its influence on multiple cell types is critical for the regulation of numerous biologic processes in the host, dysregulation of both TGFß1 expression and activity is frequently observed in cancer and contributes to various aspects of cancer progression. This review focuses on TGFß1's contribution to tumor immune suppression and escape, with emphasis on the influence of this regulatory cytokine on the differentiation and function of dendritic cells and T cells. Clinical trials targeting TGFß1 in cancer patients are also reviewed, and strategies for future therapeutic interventions that build on our current understanding of immune regulation by TGFß1 are discussed.

A model system for the study of gene expression in the undergraduate laboratory.

The flow of genetic information from DNA to RNA to protein, otherwise known as the "central dogma" of biology, is one of the most basic and overarching concepts in the biological sciences. Nevertheless, numerous studies have reported student misconceptions at the undergraduate level of this fundamental process of gene expression. This study reports on the efficacy of a model system for teaching gene expression in the undergraduate laboratory. A student-centered investigation of Tgfb1 gene expression in two murine melanoma cell lines was used to emphasize not only the process of gene expression but also various research methods for studying this phenomenon. Traditional RT-PCR, quantitative real-time RT-PCR, and flow cytometry-based in situ hybridization assays were employed to study expression of this immunosuppressive cytokine gene in the highly tumorigenic B16-F1 melanoma cell line and the poorly tumorigenic D5.1G4 melanoma cell line, both at the population and single-cell levels. A pre- and post-laboratory assessment instrument demonstrated the utility of this model system in enhancing student learning both of content related to gene expression and of research methods and data analysis skills. The pedagogical approach described in this study is therefore an effective way to improve the teaching and learning of gene expression at the undergraduate level. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):397-404, 2016.

Murine and Human Model Systems for the Study of Dendritic Cell Immunobiology.

Dendritic cells are a population of innate immune cells that possess their own effector functions as well as numerous regulatory properties that shape the activity of other innate and adaptive cells of the immune system. Following their development from either lymphoid or myeloid progenitors, the function of dendritic cells is tightly linked to their maturation and activation status. Differentiation into specialized subsets of dendritic cells also contributes to the diverse immunologic functions of these cells. Because of the key role played by dendritic cells in the regulation of both immune tolerance and activation, significant efforts have been focused on understanding dendritic cell biology. This review highlights the model systems currently available to study dendritic cell immunobiology and emphasizes the advantages and disadvantages to each system in both murine and human settings. In particular, in vitro cell culture systems involving immortalized dendritic cell lines, ex vivo systems for differentiating and expanding dendritic cells from their precursor populations, and systems for expanding, ablating, and manipulating dendritic cells in vivo are discussed. Emphasis is placed on the contribution of these systems to our current understanding of the development, function, and immunotherapeutic applications of dendritic cells, and insights into how these models might be extended in the future to answer remaining questions in the field are discussed.

Melanoma-derived factors alter the maturation and activation of differentiated tissue-resident dendritic cells.

Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)ß1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth.