Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes

Background: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. Methods: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). Results: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. Conclusions: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.publishedVersio

Evaluation of β-catenin inhibition of axitinib and nitazoxanide in human monocyte-derived dendritic cells

Modulation of β-catenin signaling has attractive therapeutic potential in cancer immunotherapy. Several studies have found that β-catenin can mediate immune evasion in cancer and promote anti-inflammatory features of antigen-presenting dendritic cells. Many small molecular compounds that inhibit Wnt/β-catenin signaling are currently in clinical development, but none have entered routine clinical use. New inhibitors of β-catenin signaling are consequently desirable. Here, we have tested, in monocyte-derived dendritic cells, the effects of two small molecular compounds, axitinib and nitazoxanide, that previously have been discovered to inhibit β-catenin signaling in colon cancer cells. Immature and lipopolysaccharide-matured dendritic cells prepared from healthy blood donor buffy coats were stimulated with 6-bromoindirubin-3′-oxime (6-BIO) to boost basal β-catenin activity, and the effects of axitinib and nitazoxanide were compared with the commercial β-catenin inhibitor ICG-001. Assays, including genome-wide RNA-sequencing, indicated that neither axitinib nor nitazoxanide demonstrated considerable β-catenin inhibition. Both compounds were found to be less toxic to monocyte-derived dendritic cells than either 6-BIO or ICG-001. Axitinib stimulated several aspects of dendritic cell function, such as IL12-p70 secretion, and counteracted IL-10 secretion, according to the present study. However, neither axitinib nor nitazoxanide were found to be efficient β-catenin inhibitors in monocyte-derived dendritic cells.publishedVersio

GM-CSF, Flt3-L and IL-4 affect viability and function of conventional dendritic cell types 1 and 2

Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.publishedVersio

Proteasome‐mediated regulation of GATA2 expression and androgen receptor transcription in benign prostate epithelial cells

GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level.publishedVersio

Lipocalin 2 expression is associated with aggressive features of endometrial cancer

Background: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. Methods: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. Results: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, β-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. Conclusion: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts

Background: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. Methods: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. Results: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Conclusions: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.publishedVersio

A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.publishedVersio

Hyperoxic Treatment Induces Mesenchymal-to-Epithelial Transition in a Rat Adenocarcinoma Model

Tumor hypoxia is relevant for tumor growth, metabolism and epithelial-to-mesenchymal transition (EMT). We report that hyperbaric oxygen (HBO) treatment induced mesenchymal-to-epithelial transition (MET) in a dimetyl-α-benzantracene induced mammary rat adenocarcinoma model, and the MET was associated with extensive coordinated gene expression changes and less aggressive tumors. One group of tumor bearing rats was exposed to HBO (2 bar, pO2 = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO2 = 0.2 bar). Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation, cell death, collagen fibrils and gene expression profile. Tumor growth was significantly reduced (∼16%) after HBO treatment compared to day 1 levels, whereas control tumors increased almost 100% in volume. Significant decreases in tumor cell proliferation, tumor blood vessels and collagen fibrils, together with an increase in cell death, are consistent with tumor growth reduction and tumor stroma influence after hyperoxic treatment. Gene expression profiling showed that HBO induced MET. In conclusion, hyperoxia induced MET with coordinated expression of gene modules involved in cell junctions and attachments together with a shift towards non-tumorigenic metabolism. This leads to more differentiated and less aggressive tumors, and indicates that oxygen per se might be an important factor in the “switches” of EMT and MET in vivo. HBO treatment also attenuated tumor growth and changed tumor stroma, by targeting the vascular system, having anti-proliferative and pro-apoptotic effects

Global profiling of histone and DNA methylation reveals epigenetic-based regulation of gene expression during epithelial to mesenchymal transition in prostate cells

<p>Abstract</p> <p>Background</p> <p>Previously we reported extensive gene expression reprogramming during epithelial to mesenchymal transition (EMT) of primary prostate cells. Here we investigated the hypothesis that specific histone and DNA methylations are involved in coordination of gene expression during EMT.</p> <p>Results</p> <p>Genome-wide profiling of histone methylations (H3K4me3 and H3K27me3) and DNA methylation (DNAMe) was applied to three cell lines at different stages of a stepwise prostate cell model involving EMT and subsequent accumulation of malignant features. Integrated analyses of epigenetic promoter modifications and gene expression changes revealed strong correlations between the dynamic changes of histone methylations and gene expression. DNA methylation was weaker associated with global gene repression, but strongly correlated to gene silencing when genes co-modified by H3K4me3 were excluded. For genes labeled with multiple epigenetic marks in their promoters, the level of transcription was associated with the net signal intensity of the activating mark H3K4me3 minus the repressive marks H3K27me3 or DNAMe, indicating that the effect on gene expression of bivalent marks (H3K4/K27me3 or H3K4me3/DNAMe) depends on relative modification intensities. Sets of genes, including epithelial cell junction and EMT associated fibroblast growth factor receptor genes, showed corresponding changes concerning epigenetic modifications and gene expression during EMT.</p> <p>Conclusions</p> <p>This work presents the first blueprint of epigenetic modifications in an epithelial cell line and the progeny that underwent EMT and shows that specific histone methylations are extensively involved in gene expression reprogramming during EMT and subsequent accumulation of malignant features. The observation that transcription activity of bivalently marked genes depends on the relative labeling intensity of individual marks provides a new view of quantitative regulation of epigenetic modification.</p

Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia

<p>Abstract</p> <p>Background</p> <p>The molecular changes <it>in vivo </it>in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response.</p> <p>Methods</p> <p>The gene expression was examined by whole genome 44 k oligo microarrays and 12 k cDNA microarrays in peripheral blood leukocytes collected from seven leukemia patients before treatment, 2–4 h and 18–24 h after start of chemotherapy and validated by real-time quantitative PCR. Statistically significantly upregulated genes were classified using gene ontology (GO) terms. Parallel samples were examined by flow cytometry for apoptosis by annexin V-binding and the expression of selected proteins were confirmed by immunoblotting.</p> <p>Results</p> <p>Significant differential modulation of 151 genes were found at 4 h after start of induction therapy with cytarabine and anthracycline, including significant overexpression of 31 genes associated with p53 regulation. Within 4 h of chemotherapy the BCL2/BAX and BCL2/PUMA ratio were attenuated in proapoptotic direction. FLT3 mutations indicated that non-responders (5/7 patients, 8 versus 49 months survival) are characterized by a unique gene response profile before and at 4 h. At 18–24 h after chemotherapy, the gene expression of p53 target genes was attenuated, while genes involved in chemoresistance, cytarabine detoxification, chemokine networks and T cell receptor were prominent. No signs of apoptosis were observed in the collected cells, suggesting the treated patients as a physiological source of pre-apoptotic cells.</p> <p>Conclusion</p> <p>Pre-apoptotic gene expression can be monitored within hours after start of chemotherapy in patients with acute myeloid leukemia, and may be useful in future determination of therapy responders. The low number of patients and the heterogeneity of acute myeloid leukemia limited the identification of gene expression predictive of therapy response. Therapy-induced gene expression reflects the complex biological processes involved in clinical cancer cell eradication and should be explored for future enhancement of therapy.</p