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Bone marrow fibrosis in myeloproliferative neoplasm (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML) is associated with poor prognosis and early treatment failure. Myelofibrosis (MF) is accompanied by reprogramming of multipotent bone marrow mesenchymal stromal cells (MSC) into osteoid and fiber-producing stromal cells. We demonstrate NRP2 and osteolineage marker NCAM1 (neural cell adhesion molecule 1) expression within the endosteal niche in normal bone marrow and aberrantly in MPN, MDS MPN/MDS overlap syndromes and AML ( n = 99), as assessed by immunohistochemistry. Increased and diffuse expression in mesenchymal stromal cells and osteoblasts correlates with high MF grade in MPN ( p < 0.05 for NRP2 and NCAM1). Single cell RNA sequencing (scRNAseq) re-analysis demonstrated NRP2 expression in endothelial cells and partial co-expression of NRP2 and NCAM1 in normal MSC and osteoblasts. Potential ligands included transforming growth factor β1 (TGFB1) from osteoblasts and megakaryocytes. Murine ThPO and JAK2 V617F myelofibrosis models showed co-expression of Nrp2 and Ncam1 in osteolineage cells, while fibrosis-promoting MSC only express Nrp2. In vitro experiments with MC3T3-E1 pre-osteoblasts and analysis of Nrp2 - / - mouse femurs suggest that Nrp2 is functionally involved in osteogenesis. In summary, NRP2 represents a potential novel druggable target in patients with myelofibrosis.

Despite remarkable advances in treating patients with metastatic melanoma, the management of melanoma brain metastases remains challenging. Recent evidence suggests that epigenetic reprogramming is an important mechanism for the adaptation of melanoma cells to the brain environment. In this study, the methylomes and transcriptomes of a cohort of matched melanoma metastases were evaluated by integrated omics data analysis. The identified 38 candidate genes displayed distinct promoter methylation and corresponding gene expression changes in intracranial compared with extracranial metastases. The 11 most promising genes were validated on protein level in both tumor and surrounding normal tissue using immunohistochemistry. In accordance with the underlying promoter methylation and gene expression changes, a significantly different protein expression was confirmed for STK10, PDXK, WDR24, CSSP1, NMB, RASL11B, phosphorylated PRKCZ, PRKCZ, and phosphorylated GRB10 in the intracranial metastases. The observed changes imply a distinct intracranial phenotype with increased protein kinase B phosphorylation and a higher frequency of proliferating cells. Knockdown of PRKCZ or GRB10 altered the expression of phosphorylated protein kinase B and decreased the viability of a brain-specific melanoma cell line. In summary, epigenetically regulated cancer-relevant alterations were identified that provide insights into the molecular mechanisms that discriminate brain metastases from other organ metastases, which could be exploited by targeting the affected signaling pathways., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2's role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The treatment of advanced renal cell carcinoma remains a challenge. To develop novel therapeutic approaches, primary cell cultures as an in vitro model are considered more representative than commercial cell lines. In this study, we analyzed the gene expression of previously established primary cell cultures of clear cell renal cell carcinoma by bulk (3'm)RNA sequencing and compared it to the tissue of origin. The objectives were the identification of dysregulated pathways under cell culture conditions. Furthermore, we assessed the suitability of primary cell cultures for studying crucial biological pathways, including hypoxia, growth receptor signaling and immune evasion. RNA sequencing of primary cell cultures of renal cell carcinoma and a following Enrichr database analysis revealed multiple dysregulated pathways under cell culture conditions. 444 genes were significantly upregulated and 888 genes downregulated compared to the tissue of origin. The upregulated genes are crucial in DNA repair, cell cycle, hypoxia and metabolic shift towards aerobic glycolysis. A downregulation was observed for genes involved in pathways of immune cell differentiation and cell adhesion. We furthermore observed that 7275 genes have a similar mRNA expression in cell cultures and in tumor tissue, including genes involved in the immune checkpoint signaling or in pathways responsible for tyrosine kinase receptor resistance. Our findings confirm that primary cell cultures are a representative tool for specified experimental approaches. The results presented in this study give further valuable insights into the complex adaptation of patient-derived cells to a new microenvironment, hypoxia and other cell culture conditions, which are often neglected in daily research, and allow new translational and therapeutic approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Simon, Esser, Ellinger, Ritter, Kristiansen, Muders, Mayr and Toma.)

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)–, receptor-interacting protein kinase 1 (RIPK1)–, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell–derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell–derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Background: In recent years, a variety of innovative therapeutics for castration-resistant prostate cancer have been developed, including novel anti-androgenic drugs, such as abiraterone or VPC-13566. Therapeutic monitoring of these pharmaceuticals is performed either by measuring PSA levels in serum or by imaging. PET using PSMA ligands labeled with Fluor-18 or Gallium-68 is the most sensitive and specific imaging modality for detection of metastases in advanced prostate cancer. To date, it remains unclear how PSMA expression is modulated by anti-hormonal treatment and how it correlates with PSA secretion., Methods: We analyzed modulation of PSMA-mRNA and protein expression, 68 Ga-PSMA uptake and regulation of PSA secretion by abiraterone or VPC-13566 in LNCaP cells in vitro., Results: We found that abiraterone and VPC-13566 upregulate PSMA protein and mRNA expression but block PSA secretion in LNCaP cells. Both anti-androgens also enhanced 68 Ga-PSMA uptake normalized by the number of cells, whereas abiraterone and VPC-13566 reduced 68 Ga-PSMA uptake in total LNCaP monolayers treated due to cell death., Conclusion: Our data indicate that PSA secretion and PSMA expression are differentially regulated upon anti-androgen treatment. This finding might be important for the interpretation of 68 Ga-PSMA PET images in monitoring therapies with abiraterone and VPC-13566 in prostate cancer patients, but needs to be validated in vivo.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Urothelial bladder cancer ranks among the 10 most frequently diagnosed cancers worldwide. In our previous study, the transmembrane protein neuropilin-2 (NRP2) emerged as a predictive marker in patients with bladder cancer. NRP2 consists of several splice variants; the most abundant of these, NRP2a and NRP2b, are reported to have different biological functions in lung cancer progression. For other cancer types, there are no published data on the role of these transcript variants in cancer progression and the clinical outcome. Here, we correlate NRP2 and its two most abundant transcript variants, NRP2A and NRP2B , with the clinical outcome using available genomic data with subsequent validation in our own cohort of patients with muscle-invasive bladder cancer. In addition to NRP2 , NRP1 and the NRP ligands PDGFC and PDGFD were studied. Only NRP2A emerged as an independent prognostic marker for shorter cancer-specific survival in muscle-invasive bladder cancer in our cohort of 102 patients who underwent radical cystectomy between 2008 and 2014 with a median follow-up time of 82 months. Additionally, we demonstrate that high messenger expression of NRP2 , NRP1 , PDGFC and PDGFD associates with a more aggressive disease (i.e., a high T stage, positive lymph node status and reduced survival).

Background: Colorectal cancer (CRC) development is a multi-step process resulting in the accumulation of genetic alterations. Despite its high incidence, there are currently no mouse models that accurately recapitulate this process and mimic sporadic CRC. We aimed to develop and characterize a genetically engineered mouse model (GEMM) of Apc/Kras/Trp53 mutant CRC, the most frequent genetic subtype of CRC., Methods: Tumors were induced in mice with conditional mutations or knockouts in Apc, Kras, and Trp53 by a segmental adeno-cre viral infection, monitored via colonoscopy and characterized on multiple levels via immunohistochemistry and next-generation sequencing., Results: The model accurately recapitulates human colorectal carcinogenesis clinically, histologically and genetically. The Trp53 R172H hotspot mutation leads to significantly increased metastatic capacity. The effects of Trp53 alterations, as well as the response to treatment of this model, are similar to human CRC. Exome sequencing revealed spontaneous protein-modifying alterations in multiple CRC-related genes and oncogenic pathways, resulting in a genetic landscape resembling human CRC., Conclusions: This model realistically mimics human CRC in many aspects, allows new insights into the role of TP53 in CRC, enables highly predictive preclinical studies and demonstrates the value of GEMMs in current translational cancer research and drug development.

Background: Prostate cancer (PCa) is the most prevalent solid cancer among men in Western Countries. The clinical behavior of localized PCa is highly variable. Some cancers are aggressive leading to death, while others can even be monitored safely. Hence, there is a high clinical need for precise biomarkers for identification of aggressive disease in addition to established clinical parameters., Objective: To develop an RNA expression-based score for the prediction of PCa prognosis that facilitates clinical decision making., Design, Setting, and Participants: We assessed 233 tissue specimens of PCa patients with long-term follow-up data from fresh-frozen radical prostatectomies (RPs), from formalin-fixed and paraffin-embedded RP specimens and biopsies by transcriptome-wide next-generation sequencing and customized expression microarrays., Outcome Measurements and Statistical Analysis: We applied Cox proportional hazard models to the cohorts from different platforms and specimen types. Evidence from these models was combined by fixed-effect meta-analysis to identify genes predictive of the time to death of disease (DoD). Genes were combined by a weighted median approach into a prognostic score called ProstaTrend and transferred for the prediction of biochemical recurrence (BCR) after RP in an independent cohort of The Cancer Genome Atlas (TCGA)., Results and Limitations: ProstaTrend comprising ∼1400 genes was significantly associated with DoD in the training cohort of PCa patients treated by RP (leave-one-out cross-validation, Cox regression: p=2e-09) and with BCR in the TCGA validation cohort (Cox regression: p=3e-06). The prognostic impact persisted after multivariable Cox regression analysis adjusting for Gleason grading group (GG) ≥3 and resection status (p=0.001; DoD, training cohort) and for GG≥3, pathological stage ≥T3, and resection state (p=0.037; BCR, validation cohort)., Conclusions: ProstaTrend is a transcriptome-based score that predicts DoD and BCR in cohorts of PCa patients treated with RP., Patient Summary: ProstaTrend provides molecular patient risk stratification after radical prostatectomy., (Copyright © 2020. Published by Elsevier B.V.)

Purpose: To investigate the synergistic effect of glycolysis inhibition on therapy answer to tyrosine kinase inhibitors in renal carcinoma., Methods: Primary cell cultures from 33 renal tumors including clear cell RCC (ccRCC), papillary RCC and the rare subtype chromophobe RCC as well as two metastases of ccRCC were obtained and cultivated. The patient-derived cells were verified by immunohistochemistry. CcRCC cells were further examined by exon sequencing of the von Hippel-Lindau gene (VHL) and by RNA-sequencing. Next, cell cultures of all subtypes of RCC were exposed to increasing doses of various tyrosine kinase inhibitors (axitinib, cabozantinib and pazopanib) and the glycolysis inhibitor 2-deoxy-D-glucose, alone or combined. CellTiter-Glo ® Luminescence assay and Crystal Violet staining were used to assess the inhibition of glycolysis and the viability of the cultured primary cells., Results: The cells expressed characteristic tissue markers and, in case of ccRCC cultures, the VHL status of the tumor they derived from. An upregulation of HK1, PFKP and SLC2A1 was observed, while components of the respiratory chain were downregulated, confirming a metabolic shift towards aerobic glycolysis. The tumors displayed variable individual responses for the therapeutics. All subtypes of RCC were susceptible to cabozantinib treatment indicated by decreased proliferation. Adding 2-deoxy-D-glucose to tyrosine kinase inhibitors decreased ATP production and increased the susceptibility of ccRCC to pazopanib treatment., Conclusion: This study presents a valuable tool to cultivate even uncommon and rare renal cancer subtypes and allows testing of targeted therapies as a personalized approach as well as testing new therapies such as glycolysis inhibition in an in vitro model.

Adenosquamous carcinoma of the pancreas (ASCAP) is characterized by conventional pancreatic ductal adenocarcinoma (PDAC) and squamous carcinoma components with at least 30% of the tumour showing squamous differentiation. To get further insight into the histogenesis of these lesions, we analysed the cellular organization of ASCAP compared to PDACs. Using Immunohistochemistry and triple immunofluorescence labelling studies for keratins, p63, p40, MUC1, MUC2, MUC5AC, Ki67, and EGFR we demonstrate that many ASCAPs contain a transitional zone between the K8/18-positive adenocarcinomatous component and the p63+ /p40+ /K5/K14+ squamous component initiated by the expression of p63 in K8/18+ adenocarcinomatous cells and the appearance of basally located p63+ K5/14+ cells. p63+ K5/14+ cells give rise to fully developed squamous differentiation. Notably, 25% of conventional PDACs without histologically recognizable squamous component contain foci of p63+ p40+ and K5/14+ cells similar to the transitional zone. Our data provide evidence that the squamous carcinoma components of ASCAPs originate from pre-existing PDAC via transdifferentiation of keratin K8/18-positive glandular cells to p63-, p40-, and keratin K5/14-positive squamous carcinoma cells supporting the squamous metaplasia hypothesis. Thus our findings provide new evidence about the cellular process behind squamous differentiation in ASCAPs.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA ( TAPIR-1 and -2 ) . To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA -damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa., Competing Interests: We would like to disclose an application for patent protection of identified biomarkers by the following authors: K.R., S.F., S.C., M.F., J.H., M.W. and F.H.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFβ1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFβ1, associate with TGFβ receptors, and enhance TGFβ1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2., (Copyright © 2020 Schulz, Gorodetska, Behrendt, Fuessel, Erdmann, Foerster, Datta, Mayr, Dubrovska and Muders.)

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man 1-3 GlcNAc 2 Fuc 0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man 2-3 GlcNAc 2 Fuc 1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-β-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with new treatment options urgently needed. Neuropilins-1/-2 (NRP1, NRP2) are receptors for semaphorins and angiogenic growth factors, while the GAIP interacting protein C-terminus 1 (GIPC1, aka Synectin) interacts with the neuropilins. They are overexpressed in PDAC and associated with poor survival as well as tumor-promoting activities. Thus, neuropilin and/or GIPC1 silencing may inhibit PDAC growth. In this study, we directly compare the various tumor-inhibitory effects of transient RNAi-mediated depletion of NRP1, NRP2 and GIPC1, alone or in combination, in a set of cell lines with different expression levels. Inhibition of anchorage-dependent and -independent proliferation, colony formation and cell migration, alterations of 3D-spheroid size and shape as well as retardation of cell cycle and induction of apoptosis have been analyzed and found to vary between cell lines. The observed effects are independent of initial expression levels. Knocking down NRP1, NRP2, and GIPC1 alone demonstrates significant effects. Only small additive effects upon combined knockdown and no counter-upregulation of the respective other genes could be detected. Making the study more translational, we show that systemic treatment of PDAC xenograft-bearing mice with polymeric nanoparticles for delivery of specific siRNAs results in tumor inhibition, reduces proliferation, and induces apoptosis. In conclusion, NRP and GIPC1 inhibition emerges as a promising avenue in PDAC treatment due to pleiotropic tumor-inhibitory effects.

Purpose: PIWI-interacting RNAs (piRNAs) have been suggested to serve as biomarkers in cancer. In this study, we validated the expression profile of two piRNAs derived from mitochondria, piR-34536 and piR-51810, in tissue and serum of a cohort of clear cell renal cell carcinoma (ccRCC) patients., Methods: Tissue and serum samples of patients with ccRCC were collected prospectively in our biobank. Total RNA was isolated from 118 ccRCC tissues, 75 normal renal tissues as well as 30 serum samples from patients with ccRCC, and 15 serum samples from patients with non-malignant diseases. The expression of piRNAs was determined using quantitative real-time PCR., Results: Both piR-34536 and piR-51810 were downregulated in ccRCC compared to non-malignant renal tissue. Decreased tissue piRNA levels were significant and independent predictors of shortened progression-free, cancer-specific and overall survival of ccRCC patients. The piRNA levels in serum did not differ in ccRCC patients and control subjects., Conclusions: The expression of piR-34536 and piR-51810 in ccRCC tissues may be used as prognostic biomarkers in ccRCC.

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8 + T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM. Significance: Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg Cancer Res; 78(19); 5600-17. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Prostate cancer is the most common malignancy in men and has a high propensity to metastasize to bone. WNT5A has recently been implicated in the progression of prostate cancer, however, the receptors that mediate its effects remain unknown. Here, we identified Wnt receptors that are highly expressed in prostate cancer and investigated which of these receptors mediate the anti-tumor effects of WNT5A in prostate cancer in vitro . Extensive in vitro analyses revealed that the WNT5A receptors FZD5 and RYK mediate the anti-tumor effects of WNT5A on prostate cancer cells. Knock-down of FZD5 completely abrogated the anti-proliferative effect of WNT5A in PC3 cells. In contrast, knock-down of RYK and FZD8 did not rescue the inhibition of proliferation after WNT5A overexpression. In contrast, RYK knock-down inhibited the pro-apoptotic effect of WNT5A in PC3 cells by 60%, whereas the knock-down of either FZD5 or FZD8 further stimulated apoptosis after WNT5A overexpression (by 33% and 234%, respectively). Surface plasmon resonance analysis indicated that WNT5A has a 30% stronger binding response to FZD5 than to RYK. Further investigations using a tissue microarray revealed that expression of RYK is increased in advanced prostate cancer tumor stages, but is not associated with survival of prostate cancer patients. In contrast, patients with low local FZD5 expression, in particular in combination with low WNT5A expression, showed a longer disease-specific survival. In conclusion, WNT5A/FZD5 and WNT5A/RYK signaling are both involved in mediating the pro-apoptotic and anti-proliferative effects of WNT5A in prostate cancer., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest to disclose.

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E 2 -benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Neuropilins (NRPs) are single transmembrane receptors with short cytoplasmic tails and are dependent on receptors like VEGF receptors or Plexins for signal transduction. NRPs are known to be important in angiogenesis, lymphangiogenesis, and axon guidance. The Neuropilin-family consists of two members, Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). They are up to 44 % homologous and conserved in all vertebrates. High levels of NRP2 are found on immune cells. Current research is very limited regarding the functions of NRP2 on these cells. Recent evidence suggests that NRP2 is important for migration, antigen presentation, phagocytosis and cell-cell contact within the immune system. Additionally, posttranslational NRP2 modifications like polysialylation are crucial for the function of some immune cells. This review is an overview about expression and functions of NRP2 in the immune system., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired. As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

Evidence of sex-specific differences in renin-angiotensin-system (RAS) and arterial pressure has been shown in many mammals, including spontaneously hypertensive rats (SHRs). Although SHRs have been used extensively as a leading experimental model of hypertension, the effects of sex-specific differences in RAS on aortic function and related cardiac remodeling during aging and hypertension have not been documented in detail. We examined structural and functional changes in aorta and heart of female and male SHRs at the ages of 5, 14, 29, and 36 wk. SHRs of both sexes were hypertensive from 14 wk. Aortic endothelial dysfunction and fibrosis, left ventricular (LV) hypertrophy, and cardiac fibrosis were evident at the age of 29 wk in male SHRs but first appeared only at the age of 36 wk in female SHRs. There was a pronounced delay of matrix metalloproteinase-2 activity in the aorta and heart of female SHRs, which was associated with preservation of 40% more elastin and less extensive cardiac fibrosis than in males. At 5, 29, and 36 wk of age, female SHRs showed higher levels of aortic and myocardial AT 2 R and MasR mRNA and decreased ANG II-mediated aortic constriction. Although female SHRs had increased relaxation to AT 2 R stimulation at 5 and 29 wk compared with males, this difference disappeared at 36 wk of age. This study documents sex-specific differences in the temporal progression of aortic dysfunction and LV hypertrophy in SHRs, which are independent of arterial pressure and are apparently mediated by higher AT 2 R expression in the heart and aorta of female SHRs., (Copyright © 2017 the American Physiological Society.)

Cancer is one of the leading non-communicable diseases with highest mortality rates worldwide. About half of all cancer patients receive radiation treatment in the course of their disease. However, treatment outcome and curative potential of radiotherapy is often impeded by genetically and/or environmentally driven mechanisms of tumor radioresistance and normal tissue radiotoxicity. While nanomedicine-based tools for imaging, dosimetry and treatment are potential keys to the improvement of therapeutic efficacy and reducing side effects, radiotherapy is an established technique to eradicate the tumor cells. In order to progress the introduction of nanoparticles in radiooncology, due to the highly interdisciplinary nature, expertise in chemistry, radiobiology and translational research is needed. In this report recent insights and promising policies to design nanotechnology-based therapeutics for tumor radiosensitization will be discussed. An attempt is made to cover the entire field from preclinical development to clinical studies. Hence, this report illustrates (1) the radio- and tumor-biological rationales for combining nanostructures with radiotherapy, (2) tumor-site targeting strategies and mechanisms of cellular uptake, (3) biological response hypotheses for new nanomaterials of interest, and (4) challenges to translate the research findings into clinical trials., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12-/- mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox-/- mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox-/- mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox-/- mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19-/- bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19-/- mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

Background: Radiotherapy has a high curative potential in localized prostate cancer, however, there are still patients with locally advanced tumours who face a considerable risk of recurrence. Radiosensitization using molecular targeted drugs could help to optimize treatment for this high-risk group. The PI3K/Akt pathway is overexpressed in many prostate cancers and is correlated to radioresistance. Nelfinavir, an HIV protease inhibitor (HPI), was found to block this pathway and to radiosensitize cancer cells of different origin. This is the first study examining the effect of nelfinavir in combination with irradiation on prostate cancer cell survival in vitro as well as on growth time and local tumour control in vivo ., Methods: The in vitro effect of nelfinavir on radioresponse of PC-3 was tested by colony formation assay with 10 μM nelfinavir. In vivo , the effect of nelfinavir alone and in combination with irradiation was tested in nude mice carrying PC-3 xenografts. For evaluating tumour growth time, mice were treated with 80 mg nelfinavir/kg body weight, daily at 5 days per week over 6 weeks. Simultaneous irradiation with 30 fractions and total doses between 30 and 120 Gy was applied to calculate local tumour control for day 180 after treatment., Results: Nelfinavir inhibited Akt phosphorylation at Ser473 and showed a minor but significant effect on clonogenic cell survival in vitro with slightly higher cell survival rates after combined treatment. The treatment of PC-3 xenografts with nelfinavir alone led to no significant increase of tumour growth time and no improvement of local tumour control., Conclusions: Despite promising growth delay effects of nelfinavir in other tumour models and first clinical applications of this drug as anti-cancer agent, PC-3 prostate cancer cells express no or only minor sensitivity to nelfinavir treatment alone and no radiosensitizing effect in vitro or in vivo .

Purpose: Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for "brain-specific" therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases., Experimental Design and Results: Buparlisib inhibited AKT activity, decreased proliferation, and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. In addition, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned medium. The growth of tumors induced by injecting human BRAF- and NRAS-mutant metastatic melanoma cells into the brain of mice was significantly inhibited by buparlisib., Conclusions: These results emphasize the value of targeting the PI3K pathway as a strategy to develop drugs for melanoma brain metastases. Clin Cancer Res; 22(23); 5818-28. ©2016 AACR., (©2016 American Association for Cancer Research.)

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17β-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)-protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer.

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat- and FYVE-domain-containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies., (©2015 American Association for Cancer Research.)

Objective: Endothelial cell activation by tumor necrosis factor (TNF) and associated leukocyte infiltration are hallmarks of vasculitis. The aim of this study was to investigate the potential role of the cellular stress-associated endothelial X-box binding protein 1 (XBP-1) transcription factor in TNF-induced endothelial cell inflammation and vasculitis., Methods: Mice with an endothelial cell-specific XBP-1 deficiency were used in a modified local Shwartzman reaction (LSR) model of TNF-induced small vessel vasculitis. To address the contribution of XBP-1 to the TNF-mediated inflammatory response in endothelial cells, we examined the activation of XBP-1 expression by TNF as well as the effect of XBP-1 knockdown in endothelial cells on TNF-induced signaling, proinflammatory gene expression, and leukocyte-endothelial cell adhesion., Results: The active spliced form of XBP-1 in endothelial cells was triggered by TNF. In addition, endothelial XBP-1 contributed to the sustained TNF-triggered NF-κB-dependent transcriptional activation of proinflammatory molecules, which was associated with leukocyte-endothelial cell adhesion. In the LSR model, endothelial cell-specific XBP-1-deficient mice displayed significantly less vascular damage, accompanied by reduced perivascular neutrophil infiltration, as compared with wild-type mice., Conclusion: Endothelial XBP-1 is activated by TNF and regulates leukocyte-endothelial cell adhesion in vitro as well as neutrophil infiltration and vascular damage in murine vasculitis., (© 2015, American College of Rheumatology.)

Disseminated tumor cells require a special microenvironment to form metastases. This metastatic niche is organ specific and forms prior to the establishment of visible metastases. The niche is characterized by vascular remodeling and bone marrow-derived cells which have migrated into it. Studies by other groups and our own results have already shown that intranodal lymphangiogenesis is an important prerequisite for regional lymph node metastases in rectal cancer patients, and can be used as a prognostic marker for progression-free survival. Niche cells such as endothelia secrete factors that attract tumor and bone marrow-derived cells. CXCL12 is one of these factors. CXCL12 activates the CXCR4 chemokine axis and induces migration along its gradient. Several factors, such as hypoxia, have been described to regulate CXCR4 function and surface expression on tumor cells. Low molecular weight agents have been used to block CXCR4 activation. This review focuses on the function and regulation of CXCR4 and its ligand CXCL12 in metastases formation. It also discusses potential options for therapeutic blockage.

Similar to normal tissue, many tumors have a hierarchical organization where tumorigenic cancer stem cells (CSCs) differentiate into non-tumorigenic progenies. A host of studies have demonstrated that although CSCs and their non-tumorigenic progenies within the same clone can share common genotype, they display different epigenetic profiles that results in changes of multiple signaling pathways. Many of these pathways confer cell adaptation to the microenvironmental stresses including inflammation, hypoxia, low pH, shortage in nutrients and anti-cancer therapies. Treatment strategies based on combination of conventional therapies targeting bulk tumor cells and CSC-specific pathway inhibition bear a promise to improve cancer cure compared to monotherapies. In this review we describe the mechanisms of CSC-related therapy resistance including drug efflux by ABC transporters, activation of aldehyde dehydrogenase and developmental pathways, enhanced DNA damage response, autophagy and microenvironmental conditions, and discuss possible therapeutic strategies for improving cancer treatment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Prostate cancer is the most frequent malignancy in men, and a major cause of prostate cancer-related death is attributable to bone metastases. WNT5A is known to influence the clinical outcome of various cancer types, including prostate cancer, but the exact mechanisms remain unknown. The goal of this study was to assess the relevance of WNT5A for the development and progression of prostate cancer. WNT5A expression was determined in a cDNA and tissue microarray of primary tumor samples in well-defined cohorts of patients with prostate cancer. Compared with benign prostate tissue, the expression of WNT5A and its receptor Frizzled-5 was higher in prostate cancer, and patients with a WNT5A expression above the median had a higher probability of survival after 10 years. Using different osteotropic human prostate cancer cell lines, the influence of WNT5A overexpression and knock-down on proliferation, migration, and apoptosis was assessed. In vitro, WNT5A overexpression induced prostate cancer cell apoptosis and reduced proliferation and migration, whereas WNT5A knock-down showed opposite effects. In vivo, different xenograft models were used to determine the effects of WNT5A on tumor growth. Local tumor growth and tumor growth in the bone microenvironment was considerably diminished after WNT5A overexpression in PC3 cells. WNT5A exhibits antitumor effects in prostate cancer cells and may be suitable as a prognostic marker and therapeutic target for prostate cancer and associated skeletal metastases., (© 2014 American Society for Bone and Mineral Research.)

The standard treatment for invasive bladder cancer is radical cystectomy. In selected patients, bladder-sparing therapy can be performed by transurethral resection (TURBT) and radio-chemotherapy (RCT) or radiotherapy (RT). Our published in vitro data suggest that the Neuropilin-2 (NRP2)/VEGF-C axis plays a role in therapy resistance. Therefore, we studied the prognostic impact of NRP2 and VEGF-C in 247 bladder cancer patients (cN0M0) treated with TURBT and RCT (n = 198) or RT (n = 49) and a follow-up time up to 15 years. A tissue microarray was analyzed by immunohistochemistry. NRP2 expression emerged as a prognostic factor in overall survival (OS; HR: 3.42; 95% CI: 1.48 - 7.86; p = 0.004) and was associated with a 3.85-fold increased risk of an early cancer specific death (95% CI: 0.91 - 16.24; p = 0.066) in multivariate analyses. Cancer specific survival (CSS) dropped from 166 months to 85 months when NRP2 was highly expressed (p = 0.037). Patients with high VEGF-C expression have a 2.29-fold increased risk of shorter CSS (95% CI: 1.03-5.35; p = 0.043) in univariate analysis. CSS dropped from 170 months to 88 months in the case of high VEGF-C expression (p = 0.041). Additionally, NRP2 and VEGF-C coexpression is a prognostic marker for OS in multivariate models (HR: 7.54; 95% CI: 1.57-36.23; p = 0.012). Stratification for muscle invasiveness (T1 vs. T2-T4) confirmed the prognostic role of NRP2 and NRP2/VEGF-C co-expression in patients with T2-T4 but also with high risk T1 disease. In conclusion, immunohistochemistry for NRP2 and VEGF-C has been determined to predict therapy outcome in bladder cancer patients prior to TURBT and RCT., (© 2014 UICC.)

Unlabelled: The low-grade inflammatory state present in obesity contributes to obesity-related metabolic dysregulation, including nonalcoholic steatohepatitis (NASH) and insulin resistance. Intercellular interactions between immune cells or between immune cells and hepatic parenchymal cells contribute to the exacerbation of liver inflammation and steatosis in obesity. The costimulatory molecules, B7.1 and B7.2, are important regulators of cell-cell interactions in several immune processes; however, the role of B7 costimulation in obesity-related liver inflammation is unknown. Here, diet-induced obesity (DIO) studies in mice with genetic inactivation of both B7.1 and B7.2 (double knockout; DKO) revealed aggravated obesity-related metabolic dysregulation, reduced insulin signalling in the liver and adipose tissue (AT), glucose intolerance, and enhanced progression to steatohepatitis resulting from B7.1/B7.2 double deficiency. The metabolic phenotype of B7.1/B7.2 double deficiency upon DIO was accompanied by increased hepatic and AT inflammation, associated with largely reduced numbers of regulatory T cells (Tregs) in these organs. In order to assess the role of B7 costimulation in DIO in a non-Treg-lacking environment, we performed antibody (Ab)-mediated inhibition of B7 molecules in wild-type mice in DIO. Antibody-blockade of both B7.1 and B7.2 improved the metabolic phenotype of DIO mice, which was linked to amelioration of hepatic steatosis and reduced inflammation in liver and AT., Conclusion: Our study demonstrates a dual role of B7 costimulation in the course of obesity-related sequelae, particularly NASH. The genetic inactivation of B7.1/B7.2 deteriorates obesity-related liver steatosis and metabolic dysregulation, likely a result of the intrinsic absence of Tregs in these mice, rendering DKO mice a novel murine model of NASH. In contrast, inhibition of B7 costimulation under conditions where Tregs are present may provide a novel therapeutic approach for obesity-related metabolic dysregulation and, especially, NASH., (© 2014 by the American Association for the Study of Liver Diseases.)

Background: The Wnt inhibitor Dickkopf-1 (DKK-1) has been linked to the progression of malignant bone disease by impairing osteoblast activity. In addition, there is increasing data to suggest direct tumor promoting effects of DKK-1. The prognostic role of DKK-1 expression in prostate cancer remains unclear., Methods: A prostate cancer tissue microarray (n = 400) was stained for DKK-1 and DKK-1 serum levels were measured in 80 patients with prostate cancer. The independent prognostic value of DKK-1 expression was assessed using multivariate analyses., Results: DKK-1 tissue expression was significantly increased in prostate cancer compared to benign disease, but was not correlated with survival. However, high DKK-1 serum levels at the time of the diagnosis were associated with a significantly shorter overall and disease-specific survival. Multivariate analyses defined high serum levels of DKK-1 as an independent prognostic marker in prostate cancer (HR 3.73; 95%CI 1.44-9.66, p = 0.007)., Conclusion: High DKK-1 serum levels are associated with a poor survival in patients with prostate cancer. In light of current clinical trials evaluating the efficacy of anti-DKK-1 antibody therapies in multiple myeloma and solid malignancies, the measurement of DKK-1 in prostate cancer may gain clinical relevance.

Purpose: Macroautophagy is a catabolic pathway that degrades cellular components through the lysosomal machinery. Cytoplasmic components are sequestered in double-membrane autophagosomes. They fuse with lysosomes where their cargo is delivered for degradation and recycling. Autophagy acts as a survival mechanism under stress by producing energy and as an intracellular quality management system by clearing damaged organelles like mitochondria and proteins. In this review, the regulation and the role of autophagy in cancer and therapy response are discussed. Furthermore, we will summarize methods for detecting autophagy in vitro and in vivo., Conclusion: During the early and late stages of cancer development, the role of autophagy differs. In the very early stages of carcinogenesis, autophagy has an important function by reducing cancer initiating genetic instability and aberrant protein aggregates as well as promoting anti-cancer immune response. In established malignant tumors autophagy confers resistance against metabolic stress caused by nutrient deprivation and the rapid proliferation of carcinoma cells. This function of autophagy is also important for radiation and chemotherapy resistance in cancer. Our laboratory has found that Neuropilin-2-induced autophagy is a potent mediator of therapy resistance in different cancer types. Autophagy not only promotes the survival of tumor cells, but also leads to autophagic cell death. During dysfunctional apoptosis this form of cell death mainly sensitizes cancer cells for therapy such as ionizing radiation. Therefore, the functions of autophagy during cancer progression and therapy are two-sided and further research is needed to understand these in more detail.

Introduction: Amino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer., Methods: Regulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates., Results: DKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment., Conclusion: DKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Understanding the molecular mechanisms promoting therapy resistance is important. Previously, we reported that VEGFC can promote cancer cell survival during stress via interaction with its receptor NRP2. While examining the molecular mechanisms involved in this survival, we performed a microarray study in which we identified two genes, WDFY1 and LAMP2, which have been suggested to function in autophagy. Our subsequent studies further confirmed the regulation of autophagy by the VEGFC-NRP2 axis in cancer during starvation- and chemotherapy-induced stress. We are currently in the process of determining the mechanism(s) through which WDFY1 and LAMP2 control autophagy; however, we did observe an increase in MTOR complex 1 (MTORC1) activity after the depletion of the VEGFC-NRP2 axis. It would therefore be interesting to study whether WDFY1 and LAMP2 can influence MTORC1 activity and regulate autophagy. Taken together, our data suggest that targeting the VEGFC-NRP2 axis in combination with chemotherapy could be an effective treatment for advanced cancers.

Objectives: Pancreatic endocrine tumors (PETs) share numerous features with gastrointestinal neuroendocrine (carcinoid) tumors. Targets of novel therapeutic strategies previously assessed in carcinoid tumors were analyzed in PETs (44 cases)., Methods: Activating mutations in EGFR, KIT, and PDGFRA and nonresponse mutations in KRAS were evaluated. Copy number of EGFR and HER-2/neu was quantified by fluorescence in situ hybridization. Expression of EGFR, PDGFRA, VEGFR1, TGFBR1, Hsp90, SSTR2A, SSTR5, IGF1R, mTOR, and MGMT was measured immunohistochemically., Results: Elevated EGFR copy number was found in 38% of cases but no KRAS nonresponse mutations. VEGFR1, TGFBR1, PDGFRA, SSTR5, SSTR2A, and IGF1R exhibited the highest levels of expression in the largest percentages of PETs.Anticancer drugs BMS-754807 (selective for IGF1R/IR), 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, targeting Hsp90), and axitinib (directed toward VEGFR1-3/PDGFRA-B/KIT) induced growth inhibition of human QGP-1 PET cells with IC50 values (nM) of 273, 723, and 743, respectively. At growth-inhibiting concentrations, BMS-754807 inhibited IGF1R phosphorylation; 17-AAG induced loss of EGFR, IGF1R, and VEGFR2; and axitinib increased p21(CDKN1A) expression without inhibiting VEGFR2 phosphorylation., Conclusions: Results encourage further research into multidrug strategies incorporating inhibitors targeting IGF1R or Hsp90 and into studies of axitinib combined with conventional chemotherapeutics toxic to tumor cells in persistent growth arrest.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies.

Purpose: Studies have shown that GIPC1/Synectin is an essential adaptor protein of receptors that play an important role in cancer progression and therapy resistance. This is the first study to explore the role of GIPC1/Synectin in radioresistance of prostate cancer and as a possible predictive marker for outcome of primary radiation therapy., Materials and Methods: The effect of RNA interference-mediated GIPC1/Synectin depletion on clonogenic cell survival after irradiation with 0, 2, 4, or 6 Gy was assayed in two different GIPC1/Synectin-expressing human prostate cancer cell lines. The clinical outcome data of 358 men who underwent radiotherapy of prostate cancer with a curative intention were analyzed retrospectively. Uni- and multivariate analysis was performed of prostate-specific antigen recurrence-free survival and overall survival in correlation with protein expression in pretreatment biopsy specimens. Protein expression was evaluated by standard immunohistochemistry methods., Results: In cell culture experiments, no change was detected in radiosensitivity after depletion of GIPC1/Synectin in GIPC1/Synectin-expressing prostate cancer cell lines. Furthermore, there was no correlation between GIPC1/Synectin expression in human pretreatment biopsy samples and overall or biochemical recurrence-free survival after radiotherapy in a retrospective analysis of the study cohort., Conclusion: Our results do not show a predictive or prognostic function of GIPC1/Synectin expression for the outcome of radiotherapy in prostate cancer. Furthermore, our in vitro results do not support a role of GIPC1 in the cellular radiation response. However, the role of GIPC1 in the progression of prostate cancer and its precursors should be subject to further research.

The Neuropilin receptors are increasingly recognized as receptors for vascular endothelial growth factors like VEGF-A and VEGF-C as well as other important growth factors like HGF and FGF in human vasculature and in tumor cells. More and more studies show an important role of Neuropilin in cancer biology suggesting that these transmembrane proteins might be an emerging target for new therapies in different subsets of cancer. Interestingly, blocking the adaptor protein GIPC1/Synectin that interacts with Neuropilin might be another interesting avenue for therapy. This review summarizes unfolding scientific data on these receptors and its interacting protein GIPC1/Synectin as molecular targets for therapy in pancreatic ductal adenocarcinoma.

Background & Aims: Inherited mutations in the BRCA2 tumor suppressor have been associated with an increased risk of pancreatic cancer. To establish the contribution of Brca2 to pancreatic cancer we developed a mouse model of pancreas-specific Brca2 inactivation. Because BRCA2-inactivating mutations cause defects in repair of DNA double-strand breaks that result in chromosomal instability, we evaluated whether Brca2 inactivation induced instability in pancreatic tissue from these mice and whether associated pancreatic tumors were hypersensitive to DNA damaging agents., Methods: We developed mouse models that combined pancreas-specific Kras activation and Trp53 deletion with Brca2 inactivation. Development of pancreatic cancer was assessed; tumors and nonmalignant tissues were analyzed for chromosomal instability and apoptosis. Cancer cell lines were evaluated for sensitivity to DNA damaging agents., Results: In the presence of disrupted Trp53, Brca2 inactivation promoted the development of premalignant lesions and pancreatic tumors that reflected the histology of human disease. Cancer cell lines derived from these tumors were hypersensitive to specific DNA damaging agents. In contrast, in the presence of KrasG12D, Brca2 inactivation promoted chromosomal instability and apoptosis and unexpectedly inhibited growth of premalignant lesions and tumors., Conclusions: Trp53 signaling must be modified before inactivation of the Brca2 wild-type allele, irrespective of Kras status, for Brca2-deficient cells to form tumors., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)