Despite general acceptance of the link between chronic inflammation and malignancy
April 24, 2017
Despite general acceptance of the link between chronic inflammation and malignancy the precise molecular mechanisms underlying the cancer-promoting effects of inflammation remain undefined. and and Fig. S4). To confirm the HT sequencing results we tested the NF-κB-activating ability of 12 putative NASPs by cloning them into the initial lentiviral expression vector transducing the constructs into 293-NFκB-GFP cells and NVP-BKM120 determining the percentage of GFP-positive cells 48 h after transduction. All twelve individually tested NASPs showed strong GFP reporter induction in a NVP-BKM120 large proportion of cells thus confirming their NF-κB-inducing activity and were utilized for further functional screening (Fig. 2 and and Fig. S5). It is noteworthy that this same NASPs expressed without a leucine zipper domain name or without a SEAP-derived signaling peptide completely lacked the ability to activate NF-κB reporters thus indicating the importance of dimerization or trimerization as well as ER targeting for their functionality (Fig. 2and and and = 8 mice per group). By 10 d postinjection the majority of animals that were injected with cells cotransduced with H-RasV12 and NASP expression constructs had developed large tumors requiring euthanasia due to tumor burden according to Institutional Animal Care and Use Committee regulations. The frequency of tumor formation by day 10 postinjection was 100% 87.5% and 62.5% in groups of animals coexpressing H-RasV12 with ApoF PLP and IL-27-derived NASPs respectively. In contrast 0 (0/8) of the control mice that were NVP-BKM120 injected with REF52 cells transduced with NASP expression constructs alone or with the vacant lentiviral expression vector designed any visible tumors during the same period. These results confirm that cells showing transformed nonsenescent phenotypes following coexpression of H-RasV12 and NASPs are truly transformed with potent tumor-forming capacity. Given the fact that p53 activity is the major mechanism that prevents NVP-BKM120 oncogenic Ras-induced transformation of rodent fibroblasts (inducing senescence instead) we anticipated that suppression of p53 activity caused by NASP expression might underlie the observed ability of NASPs to promote transformation in cooperation with Ras. To test this NVP-BKM120 hypothesis we assessed the effect of NASP expression on p53 activity induced by doxorubicin a DNA-damaging chemotherapeutic agent that is a known potent activator of p53. REF52 cells stably expressing individual NASPs were treated with doxorubicin for 18 h and induction of p21 was assessed. For all those eight tested NASPs doxorubicin-induced expression of p21 a well-known p53-dependent phenomenon was reduced by NASP expression (Fig. 6infection hepatocellular malignancy associated with chronic hepatitis virus contamination bladder malignancy associated with contamination and lung malignancy associated with contamination (8). Epidemiological data show that over 20% of the mortality in malignancy patients is linked to underlying chronic infections and inflammatory responses. Indeed given the fact that inflammatory components constitute approximately half of the tumor microenvironment (2) it is likely that inflammatory cells and cytokines present in the tumor milieu might contribute to tumor initiation and progression. Additional support for any mechanistic link between malignancy and inflammation is provided by the fact that this NVP-BKM120 NF-κB pathway is usually constitutively activated in many types of malignancy and is also a central regulator of immune responses that is activated by proinflammatory cytokines and microbial components during infections (10 11 Constitutively activated NF-κB might promote tumorigenesis through its known ability to induce expression of antiapoptotic factors (35) and positive regulators of proliferation such as cytokines and growth factors (36). In addition NF-κB may promote tumor angiogenesis and invasiveness (37). However whether constitutive NF-κB activation is usually a Akt2 primary contributor to tumor development or a byproduct of the inflammatory milieu surrounding tumors has not been definitively answered. Therefore this study aimed to determine whether constitutive activation of NF-κB directly promotes malignant transformation. To reach this goal we designed a functional screen to identify genetic elements capable of inducing stable constitutive NF-κB activity in cells (NF-κB-activating genetic elements NASPs). We hypothesized that use of a cell-based phenotypic readout for the screen would allow us to isolate.