Erika Bajna

Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.

Highlights ► Comparative oncological trials could speed up the development of novel therapies. ► Modeling revealed that human and canine ErbB-1 and ErbB-2 molecules are highly conserved. ► The canine ErbB molecules harbour epitopes for cetuximab and trastuzumab antibodies and indeed transmitted growth inhibitory signals. ► This knowledge opens up perspectives for comparative immunotherapy trials with canine cancer patients.

Activation-Induced Cytidine Deaminase (AID)-Associated Multigene Signature to Assess Impact of AID in Etiology of Diseases with Inflammatory Component

Activation-induced cytidine deaminase (AID) is expressed in B cells within germinal centers and is critically involved in class switch recombination and somatic hypermutation of immunoglobulin loci. Functionally active AID can additionally be detected within ectopic follicular structures developed at sites of chronic inflammation. Furthermore, AID may target non-Ig genes in B- and non-B-cell background. Therefore, AID-associated effects are of increasing interest in disease areas such as allergy, inflammation, autoimmunity, and cancer. Pathway- or disease-relevant multigene signatures have attracted substantial attention for therapeutic target proposal, diagnostic tools, and monitoring of therapy response. To delineate the impact of AID in etiology of multifactorial diseases, we designed the AID-associated 25-gene signature. Chronic rhinosinusitis with nasal polyps was used as an inflammation-driven airway disease model; high levels of IgE have been previously shown to be present within polyp tissue. Expression levels of 16 genes were found to be modulated in polyps including AID, IgG and IgE mature transcripts which reflect AID activity; clustering algorithm revealed an AID-specific gene signature for the disease state with nasal polyp. Complementary, AID-positive ectopic lymphoid structures were detected within polyp tissues by in situ immunostaining. Our data demonstrate the class switch recombination and somatic hypermutation events likely taking place locally in the airways and in addition to the previously highlighted markers and/or targets as IL5 and IgE suggest novel candidate genes to be considered for treatment of nasal polyposis including among others IL13 and CD23. Thus, the algorithm presented herein including the multigene signature approach, analysis of co-regularities and creation of AID-associated functional network gives an integrated view of biological processes and might be further applied to assess role of altered AID expression in etiology of other diseases, in particular, aberrant immunity and cancer.

B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and pu200a=u200a0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.

The Sulfatase Pathway for Estrogen Formation: Targets for the Treatment and Diagnosis of Hormone-Associated Tumors

The extragonadal synthesis of biological active steroid hormones from their inactive precursors in target tissues is named “intracrinology.” Of particular importance for the progression of estrogen-dependent cancers is the in situ formation of the biological most active estrogen, 17beta-estradiol (E2). In cancer cells, conversion of inactive steroid hormone precursors to E2 is accomplished from inactive, sulfated estrogens in the “sulfatase pathway” and from androgens in the “aromatase pathway.” Here, we provide an overview about expression and function of enzymes of the “sulfatase pathway,” particularly steroid sulfatase (STS) that activates estrogens and estrogen sulfotransferase (SULT1E1) that converts active estrone (E1) and other estrogens to their inactive sulfates. High expression of STS and low expression of SULT1E1 will increase levels of active estrogens in malignant tumor cells leading to the stimulation of cell proliferation and cancer progression. Therefore, blocking the “sulfatase pathway” by STS inhibitors may offer an attractive strategy to reduce levels of active estrogens. STS inhibitors either applied in combination with aromatase inhibitors or as novel, dual aromatase-steroid sulfatase inhibiting drugs are currently under investigation. Furthermore, STS inhibitors are also suitable as enzyme–based cancer imaging agents applied in the biomedical imaging technique positron emission tomography (PET) for cancer diagnosis.

Gender-Specific Modulation of Markers for Premalignancy by Nutritional Soy and Calcium in the Mouse Colon

Sporadic colorectal cancer develops as a multistep process during decades of latency. Multiple factors, in particular nutrition, influence progression. Both nutritional calcium and soy are known to reduce sporadic cancer incidence. Soy contains high levels of phytoestrogens. Among them genistein is recognized as an antioxidant and cell-cycle inhibitor. However, timing and length of consumption of genistein as well as gender- and colon site-specific activity may result in beneficial or detrimental effects. We therefore evaluated the effect in mice of a basic AIN76A diet containing 20% soy as main protein source fed for 1 or 2 generations. In another set of animals, normal calcium levels (0.5%) were replaced by low calcium (0.04%) with or without supplementation of genistein (0.04%). Expression of the vitamin D receptor, cyclooxygenase (COX)-2, proapoptotic Bak and antiapoptotic Bcl-2 protein, as well as estrogen receptor (ER)-alpha and ER-beta mRNA were evaluated. Results were identical whether soy was fed for 1 or 2 generations. Soy decreased Bak and increased COX-2 and ER-alpha expression site-specifically in female mice. Vitamin D receptor protein was reduced only in males. In animals fed 0.04% dietary calcium, COX-2 protein was increased mainly in females, but supplementation of genistein to the diet lowered COX-2 expression significantly in both genders. Our results suggest that genistein counteracts the induction of a marker of colonic premalignancy by low nutritional calcium in both genders. However, soy itself enhances COX-2 and reduces Bak, but only in females. This suggests detrimental activity of an unknown component of soy triggered by a high-estrogen background.

The vitamin D system is deregulated in pancreatic diseases

Highlights • During PDAC development CYP24A1 levels are reduced in the endocrine islets.• During malignant transformation pancreatic ducts accumulate CYP24A1 protein.• CYP24A1 expression correlates with VDR in CP patients, but not in PDAC patients.• CYP24A1 overexpressing tumors are highly proliferative.• CaSR and VDR are co-expressed in the endocrine cells of the islets.

Specific expression of OATPs in primary small cell lung cancer (SCLC) cells as novel biomarkers for diagnosis and therapy

The expression of organic anion transporting polypeptides (OATPs) was elucidated in cell lines from small cell lung cancer (SCLC) and lung carcinoids and in paraffin-embedded samples from primary and metastatic SCLCs. We found a strong relationship between OATP expression and the origin of the cells, as cells from primary or metastatic SCLC and carcinoid tumors differ with respect to OATP levels. OATP4A1 is most prominent in non-malignant lung tissue and in all SCLC and carcinoid cell lines and tissues, OATP5A1 is most prominent in metastatic cells, and OATP6A1 is most prominent in SCLC cell lines and tumors. Treatment with topotecan, etoposide and cisplatin caused significant changes in the expression patterns of OATP4A1, OATP5A1, OATP6A1, chromogranin and synaptophysin. This effect was also evident in GLC-14 cells from an untreated SCLC patient before chemotherapy compared to GLC-16/-19 chemoresistant tumor cells from this patient after therapy. mRNA expression of OATP4A1, 5A1 and 6A1 correlates with protein expression as confirmed by quantitative microscopic image analysis and Western blots. OATPs might be novel biomarkers for tumor progression and the development of metastasis in SCLC patients.

Phylogenetic discordance of human and canine carcinoembryonic antigen (CEA, CEACAM) families, but striking identity of the CEA receptors will impact comparative oncology studies.

Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following this line, carcinoembryonic antigen (CEA, CEACAM5) could be a therapeutic target not only for human but also for canine (Canis lupus familiaris; dog) patients. CEACAM5 interacts with CEA-receptor (CEAR) in the cytoplasm of human cancer cells. Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and canine cancer. Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted to 23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a 99% amino acid similarity of human and canine CEA receptor (CEAR) within the phylogenetic tree. CEAR could be detected in four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a comprehensive interspecies target for novel cancer therapeutics.

Atopic donor status does not influence the uptake of the major grass pollen allergen, Phl p 5, by dendritic cells

Dendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming. In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1). In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes. The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays.

The prostaglandine E2 transporting organic anion transporting polypeptide OATP4A1: A potential prognostic marker in colorectal cancer?

430 Background: Organic anions transporters (OATPs) are important for tumor progression and therapeutic response by regulating cellular levels of hormones, second messenger proteins and drugs. OATP4A1 is a transporter of pro-inflammatory prostaglandin E2 and may contribute to cancer progression. Data on the expression of OATP4A1 and its clinical impact in primary colorectal cancer (CRC) is rare. Our study was designed to proof the overexpression of OATP4A1 in primary CRC. Methods: Frozen samples from 20 unselected CRC patients (pat) and five CRC cell lines were analyzed for OATP4A1 mRNA expression by real time PCR (mean level normalized to the calibrator, MNE). Immunohistochemistry was performed on paraffin- embedded tumor sections from 50 CRC pat., UICC 0-II (25/50 with subsequent relapse). An automatic quantitative image analysis program was applied to quantify OATP4A1 expression. Expression and intensity was correlated with clinical parameters and relapses. Results: Significant (p>0.05) higher levels o...