The Sleeping Beauty (SB) transposable element shows efficient transposition in human cells, and provides long-term transgene expression in preclinical animal models. Random chromosomal insertion of SB vectors represents a safety issue in human gene therapeutic applications, due to potential genotoxic effects associated with transposon integration. We investigated the transcriptional activities of SB in order to assess its potential to alter host gene expression upon integration. The untranslated regions (UTRs) of the transposon direct convergent, inward-directed transcription. Transcription from the 5'-UTR of SB is upregulated by the host-encoded factor high-mobility group 2-like 1 (HMG2L1), and requires a 65-base pair (bp) region not present in commonly used SB vectors. The SB transposase antagonizes the effect of HMG2L1, suggesting that natural transposase expression is under a negative feedback regulation. SB transposon vectors lacking the 65-bp region associated with HMG2L1-dependent upregulation exhibit benign transcriptional activities, at a level up to 100-times lower than that of the murine leukemia virus (MLV) long terminal repeat (LTR). Incorporation of chicken beta-globin HS4 insulator sequences in SB-based vectors reduces the transactivation of model promoters by transposon-borne enhancers, and thus may lower the risk of transcriptional activation of host genes situated close to a transposon insertion site.

Identifying hyperactive kinases in cancer is crucial for individualized treatment with specific inhibitors. Kinase activity can be discerned from global protein phosphorylation profiles obtained with mass spectrometry‐based phosphoproteomics. A major challenge is to relate such profiles to specific hyperactive kinases fueling growth/progression of individual tumors. Hitherto, the focus has been on phosphorylation of either kinases or their substrates. Here, we combined label‐free kinase‐centric and substrate‐centric information in an Integrative Inferred Kinase Activity ( INKA ) analysis. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase–substrate networks in a single biological sample. To demonstrate utility, we analyzed (i) cancer cell lines with known oncogenes, (ii) cell lines in a differential setting (wild‐type versus mutant, +/− drug), (iii) pre‐ and on‐treatment tumor needle biopsies, (iv) cancer cell panel with available drug sensitivity data, and (v) patient‐derived tumor xenografts with INKA ‐guided drug selection and testing. These analyses show superior performance of INKA over its components and substrate‐based single‐sample tool KARP , and underscore target potential of high‐ranking kinases, encouraging further exploration of INKA 's functional and clinical value.

The antioxidant enzyme peroxiredoxin 6 (Prdx6) is a key regulator of the cellular redox balance, particularly under stress conditions. We identified Prdx6 as an important player in different phases of skin carcinogenesis. Loss of Prdx6 in mice enhanced the susceptibility to skin tumorigenesis, whereas overexpression of Prdx6 in keratinocytes of transgenic mice had the opposite effect. The tumor-preventive effect of Prdx6, which was observed in a human papilloma virus 8-induced and a chemically induced tumor model, was not due to alterations in keratinocyte proliferation, apoptosis, or in the inflammatory response. Rather, endogenous and overexpressed Prdx6 reduced oxidative stress as reflected by the lower levels of oxidized phospholipids in the protumorigenic skin of Prdx6 transgenic mice and the higher levels in Prdx6-knockout mice than in control animals. In contrast to its beneficial effect in tumor prevention, overexpression of Prdx6 led to an acceleration of malignant progression of existing tumors, revealing a dual function of this enzyme in the pathogenesis of skin cancer. Finally, we found strong expression of PRDX6 in keratinocytes of normal human skin and in the tumor cells of squamous cell carcinomas, indicating a role of Prdx6 in human skin carcinogenesis. Taken together, our data point to the potential usefulness of Prdx6 activators or inhibitors for controlling different stages of skin carcinogenesis. Cancer Res; 73(11); 3460-9. Ó2013 AACR.

Pharmacologic activation of the transcription factor NRF2 has been suggested to offer a strategy for cancer prevention. In this study, we present evidence from murine tumorigenesis experiments suggesting there may be limitations to this possibility, based on tumorigenic effects of Nrf2 in murine keratinocytes that have not been described previously. In this setting, Nrf2 expression conferred metabolic alterations in keratinocytes that were protumorigenic in nature, affecting enzymes involved in glutathione biosynthesis or in the oxidative pentose phosphate pathway and other NADPH-producing enzymes. Under stress conditions, coordinate increases in NADPH, purine, and glutathione levels promoted the survival of keratinocytes harboring oncogenic mutations, thereby promoting tumor development. The protumorigenic activity of Nrf2 in keratinocytes was particularly significant in a mouse model of skin tumorigenesis that did not rely upon chemical carcinogenesis. In exploring the clinical relevance of our findings, we confirm that NRF2 and protumorigenic NRF2 target genes were activated in some actinic keratoses, the major precancerous lesion in human skin. Overall, our results reveal an unexpected tumor-promoting activity of activated NRF2 during early phases of skin tumorigenesis. Cancer Res; 75(22); 4817-29. Ó2015 AACR.

No abstract

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1–9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1–E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.

Our results provide genetic evidence for a functional cross-talk of Nrf2 and NF-κB/RelA in hepatocytes, which protects the liver from necrosis, inflammation and fibrosis. Furthermore, the double mutant mice represent a valuable animal model for iHCA.

The effectiveness of photodynamic therapy (PDT) in vivo was compared between the pulsed excimer laser-pumped dye laser system (EDL) and the continuous wave (cw) argon laser-pumped dye laser system (ADL). Serial subcutaneous transplantation was used to implant thymus aplastic nude mice with different grades of malignancy of two human squamous cell carcinomas (SCCs). Forty-eight hours after i.v. injections of a hematoporphyrin derivative (Photosan 3), the animals were irradiated with either pulsed-EDL or cw-ADL laser light at a tumor depth of 4-5 mm. The irradiation data were chosen as follows: EDL and ADL wavelength 630 nm, total dose 150 J/cm2, irradiation time 27.78 min; EDL repetition rate 30 Hz, single pulse energy 3 mJ, pulse width 20 ns; ADL intensity 90 mW/cm2. The effects of PDT were studied either by long-term observation of the animals treated or by evaluation of hematoxylin-eosin and Ki-67 histological sections of tumors 48 h after treatment. The EDL system proved to be at least as efficient as the ADL system as judged by the number of complete remissions. This became particularly evident in the treatment of the lower-graded tumors, with a good response observed in both transplanted SCCs. However, the higher-graded tumors showed a better response to PDT.