Carola Heneweer

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Inhibition of IL-6 signaling significantly reduces primary tumor growth and recurrencies in orthotopic xenograft models of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human tumors, with radical surgical resection as the only curative treatment option. However, resection is only possible in a small fraction of patients, and about 80% of the patients develop recurrencies. PDAC development is facilitated by the cytokine interleukin‐6 (IL‐6), which acts via classic and trans‐signaling. Both pathways are inhibited by the anti‐IL‐6‐receptor antibody tocilizumab, whereas the fusion protein sgp130Fc specifically blocks trans‐signaling. Here, we show that conservative or adjuvant therapy with both inhibitors reduces tumor growth in an orthotopic model of human Colo357 cells in SCID/bg mice. In the conservative setting, median primary tumor weight was reduced 2.4‐fold for tocilizumab and 4.4‐fold for sgp130Fc. sgp130Fc additionally led to a decrease in microvessel density, which was not observed with tocilizumab. In the adjuvant therapeutic setting after surgical resection of the primary tumor, treatment with tocilizumab or sgp130Fc decreased the local recurrence rate from 87.5% in the control group to 62.5 or 50%, respectively. Furthermore, the median weight of the local recurrent tumors was clearly diminished, and both inhibitors reduced the number of distant metastases. A significant reduction of tumor weight and metastases—comparable to gemcitabine treatment—was also observed with both inhibitors in another model using the poorly differentiated PancTuI cells. Our findings demonstrate the inhibition of IL‐6 as a new treatment option in PDAC.

IL‐6 trans‐signaling is essential for the development of hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin‐6 (IL‐6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL‐6 classic and the IL‐6 trans‐signaling pathway. Whereas IL‐6 classic signaling is important for innate and acquired immunity, IL‐6 trans‐signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL‐6 trans‐signaling, but not IL‐6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA‐damage‐induced hepatocyte apoptosis through suppression of p53 and enhances β‐catenin activation and tumor proliferation. Second, IL‐6 trans‐signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL‐6 trans‐signaling are largely protected from tumor formation in a diethylnitrosamine/3,3′,5,5′‐tetrachloro‐1,4‐bis(pyridyloxy)benzene model of HCC. Conclusion: IL‐6 trans‐signaling, and not IL‐6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL‐6 trans‐signaling, rather than total inhibition of IL‐6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL‐6 classic signaling‐driven protective immune responses. (Hepatology 2017;65:89‐103).

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21–CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph‐node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7‐transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph‐node metastases than mock‐transfected cells. In an analysis using quantitative real‐time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate‐to‐strong immunohistochemical CCR7 expression, found in 58 of 121 well‐characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph‐node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease‐free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor‐associated lymph vessels, representing a previously unknown factor of lymphatic spread.

Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells is L1CAM dependent

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.

Increased survival rate by local release of diclofenac in a murine model of recurrent oral carcinoma

Despite aggressive treatment with radiation and combination chemotherapy following tumor resection, the 5-year survival rate for patients with head and neck cancer is at best only 50%. In this study, we examined the therapeutic potential of localized release of diclofenac from electrospun nanofibers generated from poly(D,L-lactide-co-glycolide) polymer. Diclofenac was chosen since anti-inflammatory agents that inhibit cyclooxygenase have shown great potential in their ability to directly inhibit tumor growth as well as suppress inflammation-mediated tumor growth. A mouse resection model of oral carcinoma was developed by establishing tumor growth in the oral cavity by ultrasound-guided injection of 1 million SCC-9 cells in the floor of the mouth. Following resection, mice were allocated into four groups with the following treatment: 1) no treatment, 2) implanted scaffolds without diclofenac, 3) implanted scaffolds loaded with diclofenac, and 4) diclofenac given orally. Small animal ultrasound and magnetic resonance imaging were utilized for longitudinal determination of tumor recurrence. At the end of 7 weeks following tumor resection, 33% of mice with diclofenac-loaded scaffolds had a recurrent tumor, in comparison to 90%–100% of the mice in the other three groups. At this time point, mice with diclofenac-releasing scaffolds showed 89% survival rate, while the other groups showed survival rates of 10%–25%. Immunohistochemical staining of recurrent tumors revealed a near 10-fold decrease in the proliferation marker Ki-67 in the tumors derived from mice with diclofenac-releasing scaffolds. In summary, the local application of diclofenac in an orthotopic mouse tumor resection model of oral cancer reduced tumor recurrence with significant improvement in survival over a 7-week study period following tumor resection. Local drug release of anti-inflammatory agents should be investigated as a therapeutic option in the prevention of tumor recurrence in oral squamous carcinoma.

Laser scoop desobliteration: a method for minimally invasive remote recanalization of chronically occluded superficial femoral arteries

Abstract. Stenosis and occlusion of the superficial femoral artery (SFA) are most common in arterial occlusive disease. There are numerous interventional, surgical, and combined approaches to reconstitute maximum blood supply to the lower limb; however, despite intense clinical research, the long-term success rates are still poor. We present the first results with a catheter prototype for laser-based minimal invasive endarterectomy, called laser scoop desobliteration (LSD). The tip of a glass fiber containing a catheter was modified with a spatula head design and connected to an ultraviolet laser. It was tested in cadavers fixed with the Thiel embalming technique preserving tissue consistency, flexibility, and plasticity. After longitudinal arteriotomy of the SFA, a circular dissection between media and adventitia was performed. Then the LSD catheter was inserted and propagated with a progress of 1  mm/s. Afterward, the atheroma core, which showed a plain surface without substantial attaching tissue debris, was removed. Histological examination of the vessel wall showed that the dissection was performed at the media/adventitia interface. In summary, the constructed LSD catheter allowed a rapid and easy way to perform an endarterectomy, thereby offering an innovative approach in the treatment of chronic occluded SFA.

Impact of molecular imaging in preclinical cancer research

Development and progression of any cancer disease are the result of various alterations at the cellular and molecular level. This comprises changes in expression of genes, display of surface molecules, composition of extracellular matrix, and homing of circulating cells to the tumor site. Most of these changes occur long before morphological changes can be detected by conventional methods. Molecular imaging is aimed at the non-invasive in vivo characterization and measurement of these processes to assess therapy effects more prompt than classic morphological and functional imaging can provide. Additionally, visualization of these processes would provide more precise information about the disease expansion. Beyond that, novel therapy regimens such as immunotherapies require methods for tracking the therapeutic cells. Different imaging modalities are used for these purposes, originally established in cell biology labs like fluorescence imaging (FLI), bioluminescence imaging (BLI), and photoacoustic imaging (PAI) as well as in clinical routine like magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single photon emission CT (SPECT), and ultrasound (US). Variations in endogenous tissue contrast can be utilized for certain applications, e. g. alterations in oxygen saturation lead to signal changes in PAI and dedicated MRI sequences like BOLD sequences. However, specific contrast agents need to be designed in most assignments. These molecules usually consist of a targeting moiety that binds to molecules of interest, indicates certain functional states of the tissue, or is modified by specific enzymes on the one hand. On the other hand, another moiety is needed that changes tissue contrast in order to be detected by the chosen modality, like fluorophores for FLI, microbubbles for US, e.g. gadolinium for MRI, e.g. 18F for PET and e.g. 99mTc for SPECT. These contrast agents can either be injected into the animals whereby pharmacokinetics of the molecule itself determines the imaging protocol. Alternatively, they can be used for cell labelling in order to track these cells after injection into the animals. Most of these techniques in general provide the opportunity to be translated into clinical routine. This toolbox of endogenous and exogenous contrast agents is completed by reporter gene imaging that allows detecting changes in gene expression. This technique is in most cases limited to preclinical imaging. To visualize changes of gene expression, genetic sequences are used that code for different fluorescent proteins for FLI, different luciferases for BLI, herpes simplex virus tyrosine kinase (hsv-tk) for PET or e.g. iron-binding or iron-storage proteins for MRI. These gene sequences are brought under the control of the promoter of interest. The vectors constructed this way are then either transfected into tumor cells for preclinical transplantation models of cancer or used for the generation of transgenic animals. Pancreatic ductal adenocarcinoma (PDAC) represents one of the malignancies with the poorest prognosis where incidence equals mortality. Despite considerable advances in the understanding of the molecular mechanisms involved in the carcinogenesis of PDAC during the last decade the survival of the disease was not significantly improved over the last 40 years. Therefore, novel therapeutic approaches are urgently needed. Preclinical animal imaging can provide accurate measures of tumor progression non-invasively. Molecular imaging can in addition help, a) to detect cellular and molecular processes in vivo avoiding artefacts caused by tissue collection and preservation, b) to visualize specific therapy effects on certain signalling pathways over time, and c) to reduce animal numbers needed due to its non-invasiveness. PDAC is characterized by early spreading of metastatic cells and a high rate of local and distant recurrent disease even after complete surgical removal of the primary lesion as defined by histology. One key factor for this aggressiveness seems to be due to the high susceptibility towards inflammatory signals, part of which are acting in an autocrine manner since PDAC cells have been found to frequently express simultaneously ligands and corresponding receptors. From a surgical point of view local recurrent disease as well as distant metastases (mostly in the liver) limit the success of this curative therapeutic attempt, for which less then 20% of patients are eligible. There is strong evidence that inflammation drives these sometimes rapid and fulminant recurrences. Adjuvant chemotherapy has been shown to improve the outcome yet no long-term survival is achieved. Numerous different contrast agents for all modalities are available to detect surrogate markers of inflammation, such as endothelial adhesion molecules like E-selectin or VCAM, as well as enzymatic activity e.g. of matrix metalloproteases, caspases or cathepsins. Additionally, invasion of immune cells can be monitored by reporter gene imaging. A plethora of molecular alterations are thought to be responsible for the profound chemoresistance. Besides classical hallmarks of cancer such as mutations in the K-ras oncogene and the p53 tumor suppressor, the constitutive or inducible activity of transcription factor pathways is characteristic for PDAC. The nuclear factor-κB (NF-κB) has been shown besides others to be crucial for tumor development and apoptosis resistance mechanisms in this context. We have shown that NF-κB contributes to non-apoptotic signalling of death receptors, “normally” inducing apoptosis upon death-ligand-driven activation. Thus, targeting this pathway by established recombinant inhibitors or new drugs including natural compounds with anti-inflammatory potential might have great potential for future therapeutic concepts. In summary, in vivo animal models are a key element of understanding the processes of tumor development and progression as well as of validating novel strategies for tumor therapies. Clinically adapted animal models allow distinguishing between adjuvant, palliative and neo-adjuvant concepts. Preclinical animal imaging and especially molecular imaging can help to visualize molecular and cellular processes during the course of the disease non-invasively in vivo, and will allow a more valid validation of any novel therapeutic regime.