Development of malignant tumours is in part characterized by the ability of a tumour cell to overcome cell-cell adhesion and to invade surrounding tissue. E-cadherin is the main adhesion molecule of epithelia, and it has been implicated in carcinogenesis because it is frequently lost in human epithelial cancers. Re-establishing the functional cadherin complex in tumour cell lines results in a reversion from an invasive to a benign epithelial phenotype. However, it remained unresolved whether the loss of E-cadherin-mediated cell adhesion was a cause or a consequence of tumour progression in vivo. Here we report that the loss of E-cadherin expression coincides with the transition from well differentiated adenoma to invasive carcinoma in a transgenic mouse model of pancreatic beta-cell carcinogenesis (Rip1Tag2). Intercrossing Rip1Tag2 mice with transgenic mice that maintain E-cadherin expression in beta-tumour cells results in arrest of tumour development at the adenoma stage, whereas expression of a dominant-negative form of E-cadherin induces early invasion and metastasis. The results demonstrate that loss of E-cadherin-mediated cell adhesion is one rate-limiting step in the progression from adenoma to carcinoma.

Background-In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response. Methods and Results-Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we show that overexpression of PON2 markedly reduced ROS, whereas its knockdown increased ROS levels significantly. Using microscopic and biochemical methods, we found PON2 mainly in the nuclear membrane and endoplasmic reticulum. Furthermore, PON2 expression was induced at both the promoter and protein levels by endoplasmic reticulum stress pathway unfolded protein response. This pathway may promote both apoptotic and survival mechanisms. Functionally, PON2 reduced unfolded protein response-accompanying oxidative stress and unfolded protein response-derived caspase activation. Conclusion-We suggest that PON2 represents an endogenous defense mechanism against vascular oxidative stress and unfolded protein response-induced cell death, thereby contributing to the prevention of atherosclerosis. (Circulation.

Immune evasion in the tumor microenvironment (TME) is a crucial barrier for effective cancer therapy, and plasticity of innate immune cells may contribute to failures of targeted immunotherapies. Here, we show that rivaroxaban, a direct inhibitor of activated coagulation factor X (FX), promotes antitumor immunity by enhancing infiltration of dendritic cells and cytotoxic T cells at the tumor site. Profiling FX expression in the TME identifies monocytes and macrophages as crucial sources of extravascular FX. By generating mice with immune cells lacking the ability to produce FX, we show that myeloid cell–derived FX plays a pivotal role in promoting tumor immune evasion. In mouse models of cancer, we report that the efficacy of rivaroxaban is comparable with anti–programmed cell death ligand 1 (PD-L1) therapy and that rivaroxaban synergizes with anti–PD-L1 in improving antitumor immunity. Mechanistically, we demonstrate that FXa promotes immune evasion by signaling through protease-activated receptor 2 and that rivaroxaban specifically targets this cell-autonomous signaling pathway to reprogram tumor-associated macrophages. Collectively, our results have uncovered the importance of FX produced in the TME as a regulator of immune cell activation and suggest translational potential of direct oral anticoagulants to remove persisting roadblocks for immunotherapy and provide extravascular benefits in other diseases.

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate.

Adenovirus type 12 (Adl2) is oncogenic in neonate hamsters (1, 2), and the viral genome can chromosomally integrate into the genome of hamster cells (3-7). Adl2 DNA insertion is not site-specific; transcriptionally active cellular DNA sequences seem to be preferred targets (8, 9). The molecular mechanism of Adl2 DNA insertional recombination is akin to nonhomologous recombination, as studied in a cell-free system (10, 11). We have pursued the possibility that foreign (Adl2) DNA insertion contributes to the process of tumorigenic transformation by Adl2. The conventional notion of insertional mutagenesis disrupting cellular DNA at the immediate locus of foreign DNA integration has been extended to the concept that cellular gene activity could be affected at many different cellular sites close to or remote from the locus offoreign DNA insertion due to changes in DNA methylation patterns (5, 6, 12).In Adl2-transformed hamster cells, in Adl2-induced hamster tumor cells, and in hamster cells carrying integrated Adl2The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5515 genomes, integrated plasmid, or bacteriophage A DNA without transformed phenotype, the methylation of several cellular DNA segments is markedly enhanced in comparison with BHK21 or primary hamster cells. The randomly selected hamster cellular DNA probes used in methylation analyses have been localized by fluorescent in situ hybridization (FISH) to different hamster chromosomes that do not carry Adl2 DNA. Thus, integration of foreign DNA in hamster cells exerts a distinct trans effect on the methylation of several cellular DNA segments located on different chromosomes.

As in the development of many human cancers, in a transgenic mouse model of beta-cell carcinogenesis (Rip1Tag2), expression of neural cell adhesion molecule (NCAM) changes from the 120-kDa isoform in normal tissue to the 140/180-kDa isoforms in tumors. NCAM-deficient RiplTag2 mice, generated by crossing Rip1Tag2 mice with NCAM knockout mice, develop metastases, a tumor stage that is not seen in normal Rip1Tag2 mice. In contrast, overexpression of NCAM 120 in NCAM-deficient Rip1Tag2 mice prevents tumor metastasis. The results indicate that the loss of NCAM-mediated cell adhesion is one rate-limiting step in the actual metastatic dissemination of beta tumor cells.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death. Cancer is one of the most life-threatening diseases and will remain a demanding issue in healthcare given the increase in life expectancy, constantly growing population and incidence of risk-factor acquiring patients. It is well established that several cellular deregulations are required for durable malignant transformation. Among them are sustained proliferation, replicative immortality, evasion of immune destruction and escape from cell death.1 Thus, tumor cells represent complex, broadly re-organized entities such that effective therapies demand tumor-specific identification of underlying mechanisms to reveal 'weak points'. Combinations of multiple targeting strategies with moderate compound concentrations are more promising and provoke less side-effects than highdose single treatment. Moreover, besides targeting the machinery directly involved in cell death execution, the preceding signaling pathways are attractive targets as these harbor critical upstream mediators with many of them understudied. Generally, redox signaling at the mitochondria is a vital pathway relevant to this concept in cancer research, as it controls both energy metabolism and regulation of cell death. Therefore, it is not surprising that mitochondrial dysfunction relates to a plethora of diseases and that many lead compounds are currently being developed in order to manipulate this organelle.2 Importantly, via generation of reactive oxygen species (ROS) or Ca...

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. , was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.