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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. Cancer Res; 76(2); 418–28. ©2015 AACR.

Analysis of late endosomal protein PIKfyve

PDF file - 70K

Analysis of early and late endosomal proteins

Maturation of Cathepsin following depletion of NRP2 axis in Du145 and PC3 cells

Late endosomal protein Rab7 also affected following depletion of NRP2 or VEGF-C

PDF file - 1.6MB, Confocal microscopy and western blot results showing inhibition of autophagy and increase in chemotherapy-induced cancer cell death by specific inhibition of VEGF-C/NRP-2 axis

The depletion of NRP2 inhibits Rab switch that lead to an accumulation of immature autophagosome and early endosome

Autophagy_live cell

Depletion of NRP2 axis affect EGFR endocytosis

Overexpression of NRP2 or knockdown of WDFY1 in NRP2 depleted cell recovers the early endosomal structure

Supplementary figure legends

In times of high-precision radiotherapy, the accurate and precise definition of the primary tumor localization and its microscopic spread is of enormous importance. In glioblastoma, the microscopic tumor extension is uncertain and, therefore, population-based margins for Clinical Target Volume (CTV) definition are clinically used, which could either be too small—leading to increased risk of loco-regional recurrences—or too large, thus, enhancing the probability of normal tissue toxicity. Therefore, the aim of this project is to investigate an individualized definition of the CTV in preclinical glioblastoma models based on specific biological tumor characteristics. The microscopic tumor extensions of two different orthotopic brain tumor models (U87MG_mCherry; G7_mCherry) were evaluated before and during fractionated radiotherapy and correlated with corresponding histological data. Representative tumor slices were analyzed using Matrix-Assisted Laser Desorption/Ionization (MALDI) and stained for putative stem-like cell markers as well as invasion markers. The edges of the tumor are clearly shown by the MALDI segmentation via unsupervised clustering of mass spectra and are consistent with the histologically defined border in H&E staining in both models. MALDI component analysis identified specific peaks as potential markers for normal brain tissue (e.g., 1339 m/z), whereas other peaks demarcated the tumors very well (e.g., 1562 m/z for U87MG_mCherry) irrespective of treatment. MMP14 staining revealed only a few positive cells, mainly in the tumor border, which could reflect the invasive front in both models. The results of this study indicate that MALDI information correlates with microscopic tumor spread in glioblastoma models. Therefore, an individualized CTV definition based on biological tumor characteristics seems possible, whereby the visualization of tumor volume and protein heterogeneity can be potentially used to define radiotherapy-sensitive and resistant areas.

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 g...

SummaryInflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but pathways that link individual genetic alterations to microbiota-dependent inflammation remain to be identified. Here, we show that loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, renders Paneth cells sensitive to microbiota-, tumor-necrosis factor (TNF)- and receptor-interacting protein kinase 3 (RIPK3)-dependent cell death. This is associated with deficiency in Paneth cell-derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP is not sufficient to elicit intestinal inflammation, but provides susceptibility to pathobionts able to promote granulomatous ileitis in hosts deficient in XIAP, which can 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.

Solid tumors are characterized by global metabolic alterations which contribute to their growth and progression. Altered gene expression profiles and plasma lipid composition suggested a role for metabolic reprogramming in colorectal cancer (CRC) development. However, a conclusive picture of CRC-associated lipidome alterations in the tumor tissue has not emerged. Here, we determined molar abundances of 342 species from 20 lipid classes in matched biopsies of CRC and adjacent normal mucosa. We demonstrate that in contrast to previous reports, CRC shows a largely preserved lipidome composition that resembles that of normal colonic mucosa. Important exceptions include increased levels of lyso-phosphatidylinositols in CRC and reduced abundance of ether phospholipids in advanced stages of CRC. As such, our observations challenge the concept of widespread alterations in lipid metabolism in CRC and rather suggest changes in the cellular lipid profile that are limited to selected lipids involved in signaling and the scavenging of reactive oxygen species.

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and FISH images on a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.

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. Cancer Res; 73(1); 160–71. ©2012 AACR.

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. Cancer Res; 76(2); 418–28. ©2015 AACR.

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.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.

Several studies indicate that the aryl hydrocarbon receptor (AHR), which plays an important role in mediating the toxicity of many industrial chemicals, plays an important role in the physiology of female reproductive tract organs. This makes it likely that the AHR and additional components of the AHR signalling pathway are under the control of female sex steroids. In a previous study, we could already demonstrate the regulation of many members of the AHR battery by 17β-estradiol (E2) in the uterus of rats. In this study, we addressed the potential role of progesterone (P4) in this context. In a comparative approach using ovariectomized rats which were treated for 3 days with either vehicle control, E2, progesterone (P4) or the combination of both hormones in addition to sham-operated animals, we could demonstrate that in addition to E2, P4 is also an important factor in regulating AHR signalling in the rat uterus. P4 has effects similar to E2 on uterine Ahr, Arnt and Arnt2 mRNA levels, resulting in a downregulation of these genes, while the E2-mediated downregulation of key AHR response genes Cyp1a1, Gsta2 and Ugt1 is completely antagonized by P4. As with E2, P4 leads to an increase in uterine AHR levels, especially in the endometrial epithelium despite the decrease in corresponding mRNA levels. This indicates a complex gene-specific regulatory network involving E2, P4 and possibly AHR itself to maintain all components of the AHR signalling cascade at the required levels during all stages of the oestrous cycle and pregnancy.

Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples. The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed. VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity. We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation. Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2015-3304