Modeling Human Brain Tumors and the Microenvironment Using Induced Pluripotent Stem Cells.

Brain cancer is a group of diverse and rapidly growing malignancies that originate in the central nervous system (CNS) and have a poor prognosis. The complexity of brain structure and function makes brain cancer modeling extremely difficult, limiting pathological studies and therapeutic developments. Advancements in human pluripotent stem cell technology have opened a window of opportunity for brain cancer modeling, providing a wealth of customizable methods to simulate the disease in vitro. This is achieved with the advent of genome editing and genetic engineering technologies that can simulate germline and somatic mutations found in human brain tumors. This review investigates induced pluripotent stem cell (iPSC)-based approaches to model human brain cancer. The applications of iPSCs as renewable sources of individual brain cell types, brain organoids, blood-brain barrier (BBB), and brain tumor models are discussed. The brain tumor models reviewed are glioblastoma and medulloblastoma. The iPSC-derived isogenic cells and three-dimensional (3D) brain cancer organoids combined with patient-derived xenografts will enhance future compound screening and drug development for these deadly human brain cancers.

Long-Term Effects of Nanoscale Magnetite on Human Forebrain-like Tissue Development in Stem-Cell-Derived Cortical Spheroids.

The environmental nanoscale iron magnetite may contribute to the risk of developing neurodegenerative diseases. In addition, iron oxides can be used as the contrast agents in magnetic resonance imaging of neural tissues. The potential long-term impact of nanoscale iron oxides on cellular stress and neuro-inflammation remains unknown. The objective of this study is to evaluate the long-term effects of nanoscale iron oxide exposure on human pluripotent stem cell-derived cortical spheroids that mimic human forebrain-like tissue development. In particular, the cortical spheroids were treated with 8 nm and 15-20 nm magnetite at 0.023, 2.3, and 23 µg/mL for 4-30 days. The cell viability did not show significant differences among different test groups. The neuronal marker ß-tubulin III, cell proliferation marker Ki67, and antioxidant enzyme SOD2 did not show significant changes either. The molecular levels of cellular stress, inflammation, cell apoptosis, DNA damage and repair, and the reactive oxygen species (ROS) response were measured. A negative effect (i.e., increased inflammation and ROS response genes) of 8 nm iron oxide exposure and a positive effect (i.e., decreased inflammation, apoptosis, and ROS response and clean up genes) for 15-20 nm iron oxide exposure were observed. It is postulated that the intracellular iron content and the aggregation of iron oxides contribute to the observed differential response. Although our results demonstrate similar intracellular iron content for 8 nm and 15-20 nm groups, the aggregation is more severe for the 8 nm group (∼500 nm) than the 15 nm group (∼220-250 nm). Therefore, our data indicate an iron oxide aggregate size-dependent effects on cellular stress, inflammation, cell apoptosis, DNA damage, and the ROS response in the developing human forebrain-like tissue.

Advancements in Human Breast Cancer Targeted Therapy and Immunotherapy.

Human breast cancer treatment regimens have evolved greatly due to the significant advances in understanding the molecular mechanisms and pathways of the common subtypes of breast cancer. In this review, we discuss recent progress in breast cancer targeted therapy and immunotherapy as well as ongoing clinical trials. We also highlight the potential of combination therapies and personalized approaches to improve clinical outcomes. Targeted therapies have surpassed the hormone receptors and the human epidermal growth factor receptor 2 (HER2) to include many other molecules in targetable pathways such as the epidermal growth factor receptor (EGFR), poly (adenosine diphosphate-ribose) polymerase (PARP), and cyclin-dependent kinase 4/6 (CDK4/6). However, resistance to targeted therapy persists, underpinning the need for more efficacious therapies. Immunotherapy is considered a milestone in breast cancer treatments, including the engineered immune cells (CAR-T cell therapy) to better target the tumor cells, vaccines to stimulate the patient's immune system against tumor antigens, and checkpoint inhibitors (PD-1, PD-L1, and CTLA4) to block molecules that mediate immune inhibition. Targeted therapies and immunotherapy tested in breast cancer clinical trials are discussed here, with special emphasis on combinatorial approaches which are believed to maximize treatment efficacy and enhance patient survival.

Cytochrome P450-2D6: A novel biomarker in liver cancer health disparity.

Liver cancer morbidity and mortality rates differ among ethnic groups. In the United States, the burden of liver cancer in Asian Americans (AS) is higher compared to Caucasian Americans (CA). Research on liver cancer health disparities has mainly focused on environmental and socioeconomic factors yet has ignored the genotypic differences among various racial/ethnic groups. This lack of molecular level understanding has hindered the development of personalized medical approaches for liver cancer treatment. To understand the genetic heterogeneity of liver cancer between AS and CA, we performed a systematic analysis of RNA-seq data of AS and CA patients from The Cancer Genome Atlas (TCGA). We used four differential gene expression analysis packages; DESeq2, limma, edgeR, and Superdelta2, to identify the differentially expressed genes. Our analysis identified cytochrome P450-2D6 enzyme (CYP2D6) as the gene with the greatest differential expression with higher levels in AS compared to CA. To scrutinize the underlying mechanism of CYP2D6, Ingenuity Pathway Analysis (IPA) and Cytoscape were conducted and found hepatocyte nuclear factor-4α (HNF4A) and interleukin-6 (IL6) in direct association with CYP2D6. IL6 is downregulated in AS compared to CA, while HNF4A is not significantly different. Herein, we report that CYP2D6 may serve as a putative biomarker in liver cancer health disparities. Its negative association with IL6 proclaims an intricate relationship between CYP2D6 and inflammation in the ethnic differences seen in AS and CA liver cancer patients. The goal of the present study was to understand how genetic factors may contribute to the interethnic variability of liver cancer prevalence and outcomes in AS and CA patients. Identifying ethnic-specific genes may help ameliorate detection, diagnosis, surveillance, and treatments of liver cancer, as well as reduce disease-related incidence and mortality rates in the vulnerable population.

Studies of hormonal regulation, phenotype plasticity, bone metastasis, and experimental therapeutics in androgen-repressed human prostate cancer (ARCaP) model.

First established by Dr. Leland W. K. Chung's lab, the androgen-repressed prostate cancer cell (ARCaP) line is derived from the ascitic fluid of a prostate cancer (PCa) patient with widely metastatic disease. Based on its unique characteristic of growth suppression in the presence of androgen, ARCaP cell line has contributed to the research of PCa disease progression toward therapy- and castration-resistant PCa (t-CRPC). It has been widely applied in studies exploring experimental therapeutic reagents including Genistein, Vorinostat and Silibinin. ARCaP cells have showed increased metastatic potential to the bone and soft tissues. In addition, accumulating studies using ARCaP model have demonstrated the epithelial-to-mesenchymal transitional plasticity of PCa using epithelial-like ARCaPE line treated in vitro with growth factors derived from bone microenvironment. The resulting mesenchymal-like ARCaPM sub-clone derived from bone-metastasized tumor has high expression of several factors correlated with cancer metastasis, such as N-Cadherin, Vimentin, MCM3, Granzyme B, ß2-microglobulin and RANKL. In particular, the increased secretion of RANKL in ARCaPM further facilitates its capacity of inducing osteoclastogenesis at the bone microenvironment, leading to bone resorption and tumor colonization. Meanwhile, sphingosine kinase 1 (SphK1) acts as a key molecule driver in the neuroendocrine differentiation (NED) of ARCaP sublines, suggesting the unique facet of ARCaP cells for insightful studies in more malignant neuroendocrine prostate cancer (NEPC). Overall, the establishment of ARCaP line has provided a valuable model to explore the mechanisms underlying PCa progression toward metastatic t-CRPC. In this review, we will focus on the contribution of ARCaP model in PCa research covering hormone receptor activity, skeletal metastasis, plasticity of epithelial-to-mesenchymal transition (EMT) and application of therapeutic strategies.

Exposure of Human Lung Cells to Polystyrene Microplastics Significantly Retards Cell Proliferation and Triggers Morphological Changes.

Microplastics in the environment produced by decomposition of globally increasing waste plastics have become a dominant component of both water and air pollution. To examine the potential toxicological effects of microplastics on human cells, the cultured human alveolar A549 cells were exposed to polystyrene microplastics (PS-MPs) of 1 and 10 µm diameter as a model of the environmental contaminants. Both sizes caused a significant reduction in cell proliferation but exhibited little cytotoxicity, as measured by the maintenance of cell viabilities determined by trypan blue staining and by Calcein-AM staining. The cell viabilities did not drop below 93% even at concentrations of PS-MPs as high as 100 µg/mL. Despite these high viabilities, further assays revealed a population level decrease in metabolic activity parallel in time with a dramatic decrease in proliferation rate in PS-MP exposed cells. Furthermore, phase contrast imaging of live cells at 72 h revealed major changes in the morphology of cells exposed to microplastics, as well as the uptake of multiple 1 µm PS-MPs into the cells. Confocal fluorescent microscopy at 24 h of exposure confirmed the incorporation of 1 µm PS-MPs. These disturbances at the proliferative and cytoskeletal levels of human cells lead us to propose that airborne polystyrene microplastics may have toxicologic consequences. This is the first report of exposure of human cells to an environmental contaminant resulting in the dual effects of inhibition of cell proliferation and major changes in cell morphology. Our results make clear that human exposure to microplastic pollution has significant consequence and potential for harm to humans.

Upregulation of minichromosome maintenance complex component 3 during epithelial-to-mesenchymal transition in human prostate cancer.

Metastasis is often associated with epithelial-to-mesenchymal transition (EMT). To understand the molecular mechanisms of this process, we conducted proteomic analysis of androgen-repressed cancer of the prostate (ARCaP), an experimental model of metastatic human prostate cancer. The protein signatures of epithelial (ARCaPE) and mesenchymal (ARCaPM) cells were consistent with their phenotypes. Importantly, the expression of mini-chromosome maintenance 3 (MCM3) protein, a crucial subunit of DNA helicase, was significantly higher in ARCaPM cells than that of ARCaPE cells. This increased MCM3 protein expression level was verified using Western blot analysis of the ARCaP cell lineages. Furthermore, immunohistochemical analysis of MCM3 protein levels in human prostate tissue specimens showed elevated expression in bone metastasis and advanced human prostate cancer tissue samples. Subcutaneous injection experiments using ARCaPE and ARCaPM cells in a mouse model also revealed increased MCM3 protein levels in mesenchymal-derived tumors. This study identifies MCM3 as an upregulated molecule in mesenchymal phenotype of human prostate cancer cells and advanced human prostate cancer specimens, suggesting MCM3 may be a new potential drug target for prostate cancer treatment.

The Role of Interleukin-1 in Inflammatory and Malignant Human Skin Diseases and the Rationale for Targeting Interleukin-1 Alpha.

Inflammation plays a major role in the induction and progression of several skin diseases. Overexpression of the major epidermal proinflammatory cytokines interleukin (IL) 1 alpha (IL-1α) and 1 beta (IL-1ß) is positively correlated with symptom exacerbation and disease progression in psoriasis, atopic dermatitis, neutrophilic dermatoses, skin phototoxicity, and skin cancer. IL-1ß and the interleukin-1 receptor I (IL-1RI) have been used as a therapeutic target for some autoinflammatory skin diseases; yet, their system-wide effects limit their clinical usage. Based on the local effects of extracellular IL-1α and its precursor, pro-IL-1α, we hypothesize that this isoform is a promising drug target for the treatment and prevention of many skin diseases. This review provides an overview on IL-1α and IL-ß functions, and their contribution to inflammatory and malignant skin diseases. We also discuss the current treatment regimens, and ongoing clinical trials, demonstrating the potential of targeting IL-1α, and not IL-1ß, as a more effective strategy to prevent or treat the onset and progression of various skin diseases.

Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.

Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.

Evidence for a Proapoptotic Role of Matrix Metalloproteinase-26 in Human Prostate Cancer Cells and Tissues.

Matrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate cancer (ARCaP) cells, transfected with sense or anti-sense MMP-26 cDNA, are directly correlated with those of the pro-apoptotic marker Bax. Immunohistochemical staining of prostate cancer tissue samples shows similar protein expression patterns, correlating the expression levels of MMP-26 and Bax in benign, neoplastic, and invasive prostate cancer tissues. The MMP-26 protein levels were upregulated in high grade prostate intraepithelial neoplasia (HGPIN) and decreased during the course of disease progression. Further analysis using an indirect terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that many tumor cells expressing MMP-26 were undergoing apoptosis. This study showed that the high level of MMP-26 expression is positively correlated with the presence of apoptotic cells. This pro-apoptotic role of MMP-26 in human prostate cancer cells and tissues may enhance our understanding of the paradoxical roles of MMP-26 in tumor invasion and progression.

The Anti-inflammatory Role of Endometase/Matrilysin-2 in Human Prostate Cancer Cells.

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is an endopeptidase mostly produced by human carcinoma cells. While MMPs are thought to regulate the dynamics of extracellular matrix turnover, new evidence shows that these enzymes may play a critical regulatory role in inflammation. To investigate the role of MMP-26 in inflammation, three different variants of androgen repressed human prostate cancer (ARCaP) cells were investigated in the study: parental, MMP-26 sense cDNA-transfected, and MMP-26 antisense cDNA-transfected ARCaP cells. Protein lysates and RNA from control and genetically modified cells were analyzed by Western blotting and real-time reverse transcription polymerase chain reaction on arrays of genes critical to the inflammatory response. In comparison to parental controls, up-regulation of MMP-26 expression in MMP-26 sense cDNA-transfected cells resulted in a decrease in inflammatory genes expression. Conversely, inflammatory genes were up-regulated in MMP-26 antisense cDNA-transfected cells. Therefore, modulation of MMP-26 levels significantly affects the expression of inflammatory genes, suggesting an anti-inflammatory role of MMP-26. To determine a possible mechanism of action, further analysis, at both transcript and protein levels, revealed a dramatic down-regulation of interleukin-10 receptor B (IL10RB) in MMP-26 antisense cDNA-transfected cells. The low level of IL10RB was inversely correlated with matrix metalloproteinase-9 (MMP-9) expression. Collectively, our data suggest that the deficiency of MMP-26 may promote inflammation via inhibition of IL10RB-mediated signaling. These results propose a novel anti-inflammation function of MMP-26 and could provide novel molecular insight of therapeutic targeting.

Metastasis suppressors in human benign prostate, intraepithelial neoplasia, and invasive cancer: their prospects as therapeutic agents.

Despite advances in diagnosis and treatment of prostate cancer, development of metastases remains a major clinical challenge. Research efforts are dedicated to overcome this problem by understanding the molecular basis of the transition from benign cells to prostatic intraepithelial neoplasia (PIN), localized carcinoma, and metastatic cancer. Identification of proteins that inhibit dissemination of cancer cells will provide new perspectives to define novel therapeutics. Development of antimetastatic drugs that trigger or mimic the effect of metastasis suppressors represents new therapeutic approaches to improve patient survival. This review focuses on different biochemical and cellular functions of metastasis suppressors known to play a role in prostate carcinogenesis and progression. Ten putative metastasis suppressors implicated in prostate cancer are discussed. CD44s is decreased in both PIN and cancer; Drg-1, E-cadherin, KAI-1, RKIP, and SSeCKS show similar expression between benign epithelia and PIN, but are downregulated in invasive cancer; whereas, maspin, MKK4, Nm23 and PTEN are upregulated in PIN and downregulated in cancer. Moreover, the potential role of microRNA in prostate cancer progression, the understanding of the cellular distribution and localization of metastasis suppressors, their mechanism of action, their effect on prostate invasion and metastasis, and their potential use as therapeutics are addressed.

Active roles of tumor stroma in breast cancer metastasis.

Metastasis is the major cause of death for breast cancer patients. Tumors are heterogenous cellular entities composed of cancer cells and cells of the microenvironment in which they reside. A reciprocal dynamic interaction occurs between the tumor cells and their surrounding stroma under physiological and pathological conditions. This tumor-host communication interface mediates the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer at secondary site. Each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment and production of unique array of growth factors and chemokines. Stromal microenvironment may play active roles in breast cancer metastasis. Elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and improve patient outcome.

Analysis of tubulin alpha-1A/1B C-terminal tail post-translational poly-glutamylation reveals novel modification sites.

Tubulin-α(1A/1B) C-terminal tail (CTT) has seven glutamic acid residues among the last 11 amino acids of its sequence that are potential sites for glutamylation. Cleavage of C-terminal tyrosine resulting in the detyrosinated form of tubulin-α(1A/1B) is another major modification. These modifications among others bring about highly heterogeneous tubulin samples in brain cells and microtubules, play a major role in directing intracellular trafficking, microtubule dynamics, and mitotic events, and can vary depending on the cell and disease state, such as cancer and neurodegenerative disorders. Identified previously using primary mass spectrometry (MS) ions and partial Edman sequencing, tubulin-α(1A/1B) glutamylation was found exclusively on the E(445) residue. We here describe the analysis of tubulin-α(1A/1B) glutamylation and detyrosination after 2-DE separation, trypsin and proteinase K in-gel digestion, and nanoUPLC-ESI-QqTOF-MS/MS of mouse brain and bovine microtubules. Tyrosinated, detyrosinated, and Δ2-tubulin-α(1A/1B) CTTs were identified on the basis of a comparison of fragmentation patterns and retention times between endogenous and synthetic peptides. Stringent acceptance criteria were adapted for the identification of novel glutamylation sites. In addition to the previously identified site at E(445), glutamylation on mouse and bovine tubulin-α(1A/1B) CTTs was identified on E(441) and E(443) with MASCOT Expect values below 0.01. O-Methylation of glutamates was also observed.

Non-receptor tyrosine kinase 2 reaches its lowest expression levels in human breast cancer during regional nodal metastasis.

Almost half of breast Ductal Carcinoma in situ are likely to remain non threatening in situ lesions with no invasion to the surrounding stroma and no metastases. The majority of focal disruptions in myoepithelial (ME) cell layers indicative of invasion onset were found to be overlying epithelial cell clusters with no or substantially reduced estrogen receptor α (ERα) expression. Here we report the down-regulation of tyrosine kinase-2 (TYK2) and up-regulation of strumpellin expression, among other proteins in ERα(-) cells located at disrupted ME layers compared to adjacent ERα(+) cells overlying an intact myoepithelial layer. ERα(+) and ERα(-) cells were microdissected from the same in vivo human breast cancer tissues, proteins were extracted and separated utilizing Differential in-Gel Electrophoresis followed by trypsin digestion, MALDI-TOF analysis, and protein identification. Proteins expressed by ERα(-) cell clusters were found to express higher levels of strumpellin that binds to valosin-containing protein (VCP) to slow-down wound closure and promote growth; and lower levels of TYK2, a jak protein necessary for lineage specific differentiation. TYK2 levels were further analyzed by immunohistochemistry in a cohort composed of 70 patients with broad clinical characteristics. TYK2 levels were minimal in TxN1M0 breast cancers which is the stage where the initial regional lymph node metastasis is observed. Our data highlight the role of TYK2 downregulation in breast cancer cell de-differentitation and initiation of regional metastasis. In addition, the aggressiveness of the ERα(-) cell clusters compared to ERα(+) ones present in the same duct of the same patient was confirmed.

Novel stromal biomarkers in human breast cancer tissues provide evidence for the more malignant phenotype of estrogen receptor-negative tumors.

Research efforts were focused on genetic alterations in epithelial cancer cells. Epithelial-stromal interactions play a crucial role in cancer initiation, progression, invasion, angiogenesis, and metastasis; however, the active role of stroma in human breast tumorigenesis in relation to estrogen receptor (ER) status of epithelial cells has not been explored. Using proteomics and biochemical approaches, we identified two stromal proteins in ER-positive and ER-negative human breast cancer tissues that may affect malignant transformation in breast cancer. Two putative biomarkers, T-cell receptor alpha (TCR-α) and zinc finger and BRCA1-interacting protein with a KRAB domain (ZBRK1), were detected in leukocytes of ER-positive and endothelial cells of ER-negative tissues, respectively. Our data suggest an immunosuppressive role of leukocytes in invasive breast tumors, propose a multifunctional nature of ZBRK1 in estrogen receptor regulation and angiogenesis, and demonstrate the aggressiveness of ER-negative human breast carcinomas. This research project may identify new stromal drug targets for the treatment of breast cancer patients.

Protein profiling of isolated leukocytes, myofibroblasts, epithelial, Basal, and endothelial cells from normal, hyperplastic, cancerous, and inflammatory human prostate tissues.

In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prostate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.