Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells.

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study, a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells, and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56, CD16, and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure, while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-γ secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells, and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain, kidney, and lung cancer cell lines. Further NK maturation yielded CD56-ve CD16bright cells, which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs, coupled with their promising anti-tumor activity against ATRT in vitro, offer valuable insights into potential immunotherapeutic strategies for brain tumors.

Interconversion of Cancer Cells and Induced Pluripotent Stem Cells.

Cancer cells, especially cancer stem cells (CSCs), share many molecular features with induced pluripotent stem cells (iPSCs) that enable the derivation of induced pluripotent cancer cells by reprogramming malignant cells. Conversely, normal iPSCs can be converted into cancer stem-like cells with the help of tumor microenvironment components and genetic manipulation. These CSC models can be utilized in oncogenic initiation and progression studies, understanding drug resistance, and developing novel therapeutic strategies. This review summarizes the role of pluripotency factors in the stemness, tumorigenicity, and therapeutic resistance of cancer cells. Different methods to obtain iPSC-derived CSC models are described with an emphasis on exposure-based approaches. Culture in cancer cell-conditioned media or cocultures with cancer cells can convert normal iPSCs into cancer stem-like cells, aiding the examination of processes of oncogenesis. We further explored the potential of reprogramming cancer cells into cancer-iPSCs for mechanistic studies and cancer dependencies. The contributions of genetic, epigenetic, and tumor microenvironment factors can be evaluated using these models. Overall, integrating iPSC technology into cancer stem cell research holds significant promise for advancing our knowledge of cancer biology and accelerating the development of innovative and tailored therapeutic interventions.

TCGA RNA-Seq and Tumor-Infiltrating Lymphocyte Imaging Data Reveal Cold Tumor Signatures of Invasive Ductal Carcinomas and Estrogen Receptor-Positive Human Breast Tumors.

Comparative studies of immune-active hot and immune-deserted cold tumors are critical for identifying therapeutic targets and strategies to improve immunotherapy outcomes in cancer patients. Tumors with high tumor-infiltrating lymphocytes (TILs) are likely to respond to immunotherapy. We used the human breast cancer RNA-seq data from the cancer genome atlas (TCGA) and classified them into hot and cold tumors based on their lymphocyte infiltration scores. We compared the immune profiles of hot and cold tumors, their corresponding normal tissue adjacent to the tumor (NAT), and normal breast tissues from healthy individuals from the Genotype-Tissue Expression (GTEx) database. Cold tumors showed a significantly lower effector T cells, lower levels of antigen presentation, higher pro-tumorigenic M2 macrophages, and higher expression of extracellular matrix (ECM) stiffness-associated genes. Hot/cold dichotomy was further tested using TIL maps and H&E whole-slide pathology images from the cancer imaging archive (TCIA). Analysis of both datasets revealed that infiltrating ductal carcinoma and estrogen receptor ER-positive tumors were significantly associated with cold features. However, only TIL map analysis indicated lobular carcinomas as cold tumors and triple-negative breast cancers (TNBC) as hot tumors. Thus, RNA-seq data may be clinically relevant to tumor immune signatures when the results are supported by pathological evidence.

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.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration.

BACKGROUND: Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. METHODS: An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. RESULTS: Macrophage-conditioned media intensified the effects of breast cancer-adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte-breast cancer co-cultures treated with unconditioned media. CONCLUSIONS: A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression.

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.

Biogenesis of Extracellular Vesicles Produced from Human-Stem-Cell-Derived Cortical Spheroids Exposed to Iron Oxides.

Stem-cell-derived extracellular vesicles (EVs) are promising tools for therapeutic delivery and imaging in the medical research fields. EVs that arise from endosomal compartments or plasma membrane budding consist of exosomes and microvesicles, which range between 30 and 200 nm and 100-1000 nm, respectively. Iron oxide nanoparticles can be used to label stem cells or possibly EVs for magnetic resonance imaging. This could be a novel way to visualize areas in the body that are affected by neurological disorders such as stroke. Human induced pluripotent stem cells (iPSK3 cells) were plated on low-attachment plates and treated with SB431542 and LDN193189 during the first week for the induction of cortical spheroid formation and grown with fibroblast growth factor 2 and cyclopamine during the second week for the neural progenitor cell (iNPC) differentiation. iNPCs were then grown on attachment plates and treated with iron oxide (Fe3O4) nanoparticles at different sizes (8, 15, and 30 nm in diameter) and concentrations (0.1, 10, and 100 µM). The spheroids and media collected from these cultures were used for iron oxide detection as well as EV isolation and characterizations, respectively. MTT assay demonstrated that the increased size and concentration of the iron oxide nanoparticles had little effect on the metabolic activity of iNPCs. In addition, the Live/Dead assay showed high viability in all the nanoparticle treated groups and the untreated control. The EVs isolated from these culture groups were analyzed and displayed similar or higher EV counts compared with control. The observed EV size averaged 200-250 nm, and electron microscopy revealed the expected exosome morphology for EVs from all groups. RT-PCR analysis of EV biogenesis markers (CD63, CD81, Alix, TSG101, Syntenin1, ADAM10, RAB27b, and Syndecan) showed differential expression between the iron-oxide-treated cultures and nontreated cultures, as well as between adherent and nonadherent 3D cultures. Iron oxide nanoparticles were detected inside the cortical spheroid cells but not EVs by MRI. The addition of iron oxide nanoparticles does not induce significant cytotoxic effects to cortical spheroids. In addition,, nanoparticles may stimulate the biogenesis of EVs when added to cortical spheroids in vitro.

Identifying county-level factors for female breast cancer incidence rate through a large-scale population study.

Female breast cancer (FBC) incidence rate (IR) varies greatly across counties in the United States (U.S.). Factors contributing to these geographic disparities have not been fully understood at the population level. In this study, we investigated the relationships between the county-level FBC IR and a diverse set of variables in demographics, socioeconomics, life style, health care accessibility, and environment. Our study included 1,277 counties in the U.S. where the female population was 10,000 or above for at least one race/ethnicity. After controlling for the racial/ethnic and other significant factors, percent of husband-wife family households (pHWFH) for a racial/ethnic group in a county is negatively associated with FBC IR (p < 0.001). A 10% increase in married family households may lower a county's IR by 5.2 cases per 100,000 females per year. We also found that PM2.5 (fine inhalable particles with a diameter of 2.5 micrometers or less) is positively associated with FBC IR (p < 0.001). Counties with the highest level of PM2.5 have approximately 4 additional FBC new cases per 100,000 females per year than counties with the lowest level of PM2.5. Furthermore, we found that the county-level factors contributing to FBC IR vary significantly for different racial groups using race-specific models. While confirming most of the previously known patient- and neighborhood-level risk factors (such as race/ethnicity, income, and health care accessibility), our study identified two significant county-level factors contributing to the spatial disparity of FBC IR across the U.S. The newly-identified beneficial factor (marriage) and risk factor (PM2.5), together with the verified known factors, may help provide insights to officials of health departments/organizations for them to make decisions on cancer intervention strategies.

Secretome analysis reveals upregulated granzyme B in human androgen-repressed prostate cancer cells with mesenchymal and invasive phenotype.

Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity.

Immune landscape of human prostate cancer: immune evasion mechanisms and biomarkers for personalized immunotherapy.

BACKGROUND: Despite recent advances in cancer immunotherapy, the efficacy of these therapies for the treatment of human prostate cancer patients is low due to the complex immune evasion mechanisms (IEMs) of prostate cancer and the lack of predictive biomarkers for patient responses. METHODS: To understand the IEMs in prostate cancer and apply such understanding to the design of personalized immunotherapies, we analyzed the RNA-seq data for prostate adenocarcinoma from The Cancer Genome Atlas (TCGA) using a combination of biclustering, differential expression analysis, immune cell typing, and machine learning methods. RESULTS: The integrative analysis identified eight clusters with different IEM combinations and predictive biomarkers for each immune evasion cluster. Prostate tumors employ different combinations of IEMs. The majority of prostate cancer patients were identified with immunological ignorance (89.8%), upregulated cytotoxic T lymphocyte-associated protein 4 (CTLA4) (58.8%), and upregulated decoy receptor 3 (DcR3) (51.6%). Among patients with immunologic ignorance, 41.4% displayed upregulated DcR3 expression, 43.26% had upregulated CTLA4, and 11.4% had a combination of all three mechanisms. Since upregulated programmed cell death 1 (PD-1) and/or CTLA4 often co-occur with other IEMs, these results provide a plausible explanation for the failure of immune checkpoint inhibitor monotherapy for prostate cancer. CONCLUSION: These findings indicate that human prostate cancer specimens are mostly immunologically cold tumors that do not respond well to mono-immunotherapy. With such identified biomarkers, more precise treatment strategies can be developed to improve therapeutic efficacy through a greater understanding of a patient's immune evasion mechanisms.

Functionalization of Brain Region-specific Spheroids with Isogenic Microglia-like Cells.

Current brain spheroids or organoids derived from human induced pluripotent stem cells (hiPSCs) still lack a microglia component, the resident immune cells in the brain. The objective of this study is to engineer brain region-specific organoids from hiPSCs incorporated with isogenic microglia-like cells in order to enhance immune function. In this study, microglia-like cells were derived from hiPSCs using a simplified protocol with stage-wise growth factor induction, which expressed several phenotypic markers, including CD11b, IBA-1, CX3CR1, and P2RY12, and phagocytosed micron-size super-paramagnetic iron oxides. The derived cells were able to upregulate pro-inflammatory gene (TNF-α) and secrete anti-inflammatory cytokines (i.e., VEGF, TGF-ß1, and PGE2) when stimulated with amyloid ß42 oligomers, lipopolysaccharides, or dexamethasone. The derived isogenic dorsal cortical (higher expression of TBR1 and PAX6) and ventral (higher expression of NKX2.1 and PROX1) spheroids/organoids displayed action potentials and synaptic activities. Co-culturing the microglia-like cells (MG) with the dorsal (D) or ventral (V) organoids showed differential migration ability, intracellular Ca2+ signaling, and the response to pro-inflammatory stimuli (V-MG group had higher TNF-α and TREM2 expression). Transcriptome analysis exhibited 37 microglia-related genes that were differentially expressed in MG and D-MG groups. In addition, the hybrid D-MG spheroids exhibited higher levels of immunoreceptor genes in activating members, but the MG group contained higher levels for most of genes in inhibitory members (except SIGLEC5 and CD200). This study should advance our understanding of the microglia function in brain-like tissue and establish a transformative approach to modulate cellular microenvironment toward the goal of treating various neurological disorders.

Subgrouping breast cancer patients based on immune evasion mechanisms unravels a high involvement of transforming growth factor-beta and decoy receptor 3.

In the era of immunotherapy and personalized medicine, there is an urgent need for advancing the knowledge of immune evasion in different cancer types and identifying reliable biomarkers that guide both therapy selection and patient inclusion in clinical trials. Given the differential immune responses and evasion mechanisms in breast cancer, we expect to identify different breast cancer groups based on their expression of immune-related genes. For that, we used the sequential biclustering method on The Cancer Genome Atlas RNA-seq breast cancer data and identified 7 clusters. We found that 77.4% of the clustered tumor specimens evade through transforming growth factor-beta (TGF-ß) immunosuppression, 57.7% through decoy receptor 3 (DcR3) counterattack, 48.0% through cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and 34.3% through programmed cell death-1 (PD-1). TGF-ß and DcR3 are potential novel drug targets for breast cancer immunotherapy. Targeting TGF-ß and DcR3 may provide a powerful approach for treating breast cancer because 57.7% of patients overexpressed these two molecules. Furthermore, triple-negative breast cancer (TNBC) patients clustered equally into two subgroups: one with impaired antigen presentation and another with high leukocyte recruitment but four different evasion mechanisms. Thus, different TNBC patients may be treated with different immunotherapy approaches. We identified biomarkers to cluster patients into subgroups based on immune evasion mechanisms and guide the choice of immunotherapy. These findings provide a better understanding of patients' response to immunotherapies and shed light on the rational design of novel combination therapies.

Decoding Somatic Driver Gene Mutations and Affected Signaling Pathways in Human Medulloblastoma Subgroups.

Medulloblastoma is the most common malignant pediatric brain tumor. Prior studies have concentrated their efforts studying the four molecular subgroups: SHH, Wnt, group 3, and group 4. SHH and Wnt are driven by their canonical pathways. Groups 3 and 4 are highly metastatic and associated with aberrations in epigenetic regulators. Recent developments in the field have revealed that these subgroups are not as homogenous as previously believed. The objective of this study is to investigate the involvement of somatic driver gene mutations in these medulloblastoma subgroups. We obtained medulloblastoma data from the Catalogue of Somatic Mutations in Cancer (COSMIC), which contains distinct samples that were not previously studied in a large cohort. We identified somatic driver gene mutations and the signaling pathways affected by these driver genes for medulloblastoma subgroups using bioinformatics tools. We have revealed novel infrequent drivers in these subgroups that contribute to our understanding of tumor heterogeneity in medulloblastoma. Normally SHH signaling is activated in the SHH subgroup, however, we determined gain-of-function mutations in ubiquitin ligase (CUL1) that inhibit Gli-mediated transcription. This suggests a potential hindrance in SHH signaling for some patients. For group 3, gain-of-function in the inhibitor of proinflammatory cytokines (HIVEP3) suggests an immunosuppressive phenotype and thus a more hostile tumor microenvironment. Surprisingly, group 4 tumors possess mutations that may prompt the activation of Wnt signaling through gain-of-function mutations in MUC16 and PCDH9. These infrequent mutations detected in this study could be due to subclonal or spatially restricted alterations. The investigation of aberrant driver gene mutations can lead to the identification of new drug targets and a greater understanding of human medulloblastoma heterogeneity.

Proteomic characterization of paired non-malignant and malignant African-American prostate epithelial cell lines distinguishes them by structural proteins.

BACKGROUND: While many factors may contribute to the higher prostate cancer incidence and mortality experienced by African-American men compared to their counterparts, the contribution of tumor biology is underexplored due to inadequate availability of African-American patient-derived cell lines and specimens. Here, we characterize the proteomes of non-malignant RC-77 N/E and malignant RC-77 T/E prostate epithelial cell lines previously established from prostate specimens from the same African-American patient with early stage primary prostate cancer. METHODS: In this comparative proteomic analysis of RC-77 N/E and RC-77 T/E cells, differentially expressed proteins were identified and analyzed for overrepresentation of PANTHER protein classes, Gene Ontology annotations, and pathways. The enrichment of gene sets and pathway significance were assessed using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis, respectively. The gene and protein expression data of age- and stage-matched prostate cancer specimens from The Cancer Genome Atlas were analyzed. RESULTS: Structural and cytoskeletal proteins were differentially expressed and statistically overrepresented between RC-77 N/E and RC-77 T/E cells. Beta-catenin, alpha-actinin-1, and filamin-A were upregulated in the tumorigenic RC-77 T/E cells, while integrin beta-1, integrin alpha-6, caveolin-1, laminin subunit gamma-2, and CD44 antigen were downregulated. The increased protein level of beta-catenin and the reduction of caveolin-1 protein level in the tumorigenic RC-77 T/E cells mirrored the upregulation of beta-catenin mRNA and downregulation of caveolin-1 mRNA in African-American prostate cancer specimens compared to non-malignant controls. After subtracting race-specific non-malignant RNA expression, beta-catenin and caveolin-1 mRNA expression levels were higher in African-American prostate cancer specimens than in Caucasian-American specimens. The "ECM-Receptor Interaction" and "Cell Adhesion Molecules", and the "Tight Junction" and "Adherens Junction" pathways contained proteins are associated with RC-77 N/E and RC-77 T/E cells, respectively. CONCLUSIONS: Our results suggest RC-77 T/E and RC-77 N/E cell lines can be distinguished by differentially expressed structural and cytoskeletal proteins, which appeared in several pathways across multiple analyses. Our results indicate that the expression of beta-catenin and caveolin-1 may be prostate cancer- and race-specific. Although the RC-77 cell model may not be representative of all African-American prostate cancer due to tumor heterogeneity, it is a unique resource for studying prostate cancer initiation and progression.

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.

Personalized chemotherapy selection for breast cancer using gene expression profiles.

Choosing the optimal chemotherapy regimen is still an unmet medical need for breast cancer patients. In this study, we reanalyzed data from seven independent data sets with totally 1079 breast cancer patients. The patients were treated with three different types of commonly used neoadjuvant chemotherapies: anthracycline alone, anthracycline plus paclitaxel, and anthracycline plus docetaxel. We developed random forest models with variable selection using both genetic and clinical variables to predict the response of a patient using pCR (pathological complete response) as the measure of response. The models were then used to reassign an optimal regimen to each patient to maximize the chance of pCR. An independent validation was performed where each independent study was left out during model building and later used for validation. The expected pCR rates of our method are significantly higher than the rates of the best treatments for all the seven independent studies. A validation study on 21 breast cancer cell lines showed that our prediction agrees with their drug-sensitivity profiles. In conclusion, the new strategy, called PRES (Personalized REgimen Selection), may significantly increase response rates for breast cancer patients, especially those with HER2 and ER negative tumors, who will receive one of the widely-accepted chemotherapy regimens.