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.

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.

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.

c-Rel-dependent monocytes are potent immune suppressor cells in cancer.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of leukocytes that are important for tumorigenesis and tumor immunotherapy. They comprise up to 10% of leukocytes in the blood of tumor patients and their depletion may be required for successful tumor immunotherapy. However, the identity of MDSCs remains obscure, primarily due to their heterogeneity and lack of a known lineage-specific transcription factor specifying their differentiation. Using single-cell transcriptomics and gene knockout approaches, we now describe a subset of murine and human myeloid suppressor cells, named rel-dependent monocytes (rMos), which are programmed by the transcription factor c-Rel of the NF-κB family. Unlike MDSCs described previously, the c-Rel-dependent monocytes expressed a high amount of the proinflammatory cytokine IL-1ß together with a low level of suppressive molecule arginase 1. Both in vitro and in tumor-bearing mice, these c-Rel+ IL-1ßhi Arg1- monocytes promoted tumor growth by potently suppressing T cell function and showed a strong migratory phenotype, all of which were impaired by c-Rel deficiency or inhibition. Mechanistic studies revealed that c-Rel controlled the expression of monocyte signature genes through a unique transcriptional complex called the c-Rel enhanceosome, and IL-1ß-CCL2 crosstalk between tumor cells and the rel-dependent monocytes maintained the suppressive tumor microenvironment. Thus, c-Rel specifies the development of a suppressive monocyte population and could be selectively targeted for treating cancer.

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.

TIPE polarity proteins are required for mucosal deployment of T lymphocytes and mucosal defense against bacterial infection.

Mucosal surfaces are continuously exposed to, and challenged by, numerous commensal and pathogenic organisms. To guard against infections, a majority of the thymus-derived T lymphocytes are deployed at the mucosa. Although chemokines are known to be involved in the mucosal lymphocyte deployment, it is not clear whether lymphocytes enter the mucosa through directed migration or enhanced random migration. Here we report that TIPE (tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like) proteins mediate directed migration of T lymphocytes into lung mucosa, and they are crucial for mucosal immune defense against Streptococcus pneumoniae infection. Knockout of both Tnfaip8 and Tipe2, which encode polarity proteins that control the directionality of lymphocyte migration, significantly reduced the numbers of T lymphocytes in the lung of mice. Compared with wild-type mice, Tnfaip8-/- Tipe2-/- mice also developed more severe infection with more pathogens entering blood circulation upon nasal Streptococcus pneumoniae challenge. Single-cell RNA-sequencing analysis revealed that TIPE proteins selectively affected mucosal homing of a unique subpopulation of T cells, called "T cells-2", which expressed high levels of Ccr9, Tcf7, and Rag1/2 genes. TNFAIP8 and TIPE2 appeared to have overlapping functions since deficiency in both yielded the strongest phenotype. These data demonstrate that TIPE family of proteins are crucial for lung mucosal immunity. Strategies targeting TIPE proteins may help develop mucosal vaccines or treat inflammatory diseases of the lung.

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.

Decoupling tumor cell metastasis from growth by cellular pilot protein TNFAIP8.

Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers.

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.

TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling.

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.

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.

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.

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.