Sprouty is a cytoplasmic target of adenoviral E1A oncoproteins to regulate the receptor tyrosine kinase signalling pathway.

BACKGROUND: Oncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation. They are possesing a key role to favor viral replication via their interaction with multiple cellular proteins. In a yeast two-hybrid screen we have identified Sprouty1 (Spry1) as a target of adenoviral E1A Oncoproteins. Spry proteins are central and complex regulators of the receptor tyrosine kinase (RTK) signalling pathway. The deregulation of Spry family members is often associated with alterations of the RTK signalling and its downstream effectors, leading to the ERK pathway. RESULTS: Here, we confirm our yeast two-hybrid data, showing the interaction between Spry1 and E1A in GST pull-down and immunoprecipitation assays. We also demonstrated the interaction of E1A with two further Spry isoforms. Using deletion mutants we identified the N-terminus and the CR conserved region (CR) 3 of E1A- and the C-terminal half of Spry1, which contains the highly conserved Spry domain, as the essential sites for direct interaction between Spry and E1A. Immunofluorescent microscopy data revealed a co-localization of E1A(13S) with Spry1 in the cytoplasm. SRE and TRE reporter assays demonstrated that co-expression of Spry1 with E1A(13S) abolishes the inhibitory function of Spry1 in RTK signalling, which is consequently accompanied with a decrease of E1A13S-induced gene expression. CONCLUSIONS: These results establish Spry1 as a cytoplasmic localized cellular target for E1A oncoproteins to regulate the RTK signalling pathway, and consequently cellular events downstream of RTK that are essential for viral replication and transformation.

Transactivation-deficient DeltaTA-p73 acts as an oncogene.

The recently discovered p53-family member p73 displays significant homology to p53, but data from primary tumors and knockout mice argue against p73 being a classical tumor suppressor. We report on the overexpression of NH(2)-terminally truncated, transactivation-deficient p73 proteins (DeltaTA-p73) in human cancer cells. Moreover, we show that DeltaTA-p73 overexpression results in malignant transformation of NIH3T3 fibroblasts and tumor growth in nude mice, thereby providing the experimental evidence for an oncogenic function of DeltaTA-p73. Apparently, increased expression of NH(2)-terminally truncated p73 isoforms conveys the TP73 gene with oncogenic activity that appears to be actively selected for during tumor development.

Tumor-specific activation of hTERT-derived promoters by tumor suppressive E1A-mutants involves recruitment of p300/CBP/HAT and suppression of HDAC-1 and defines a combined tumor targeting and suppression system.

Adenovirus (Ad) E1A proteins are transcriptional regulators with antioncogenic but also transforming properties. We have previously shown that transformation-defective Ad5 E1A-derivatives are excellent tumor suppressors. For tumor-specific expression of the E1A-derivatives we intend to use tumor specific human telomerase reverse transcriptase (hTERT) core promoters. Here, we show that Spm2 and other E1A proteins with an intact amino terminus activated all hTERT constructs 10-20-fold in malignant tumor cells but not in primary fibroblasts, without affecting the activity of endogenous telomerase. The transcription rate in tumor cells was in the range of transcription from the SV40 promoter, which qualifies an E1A-hTERT system as a putative tumor targeting/expression system. The activation of the hTERT promoter by E1A was enhanced upon deletion of the Wilms' tumor 1 negative regulatory element and maintained high after deletion of the adjacent c-Myc-responsive E-box, demonstrating an important role of the remaining sequences that contain several Sp1-motifs. E1A-mediated hTERT activation was independent from the presence of the conserved region 3 (CR3) of E1A but dependent on E1A's binding to p300/CBP and recruitment of its histone acetyltransferase activity. Moreover, E1A-Spm2 and histone deacetylase-1 behaved as antagonists with respect to the regulation of transcription from the hTERT promoter. Overall, hTERT promoter/E1A-Spm2 systems may turn out to be excellent tools for transcriptionally targeted anticancer gene therapy.

A point mutation in the first splice donor leads to reduced oncogenic properties of the adenovirus serotype 12 E1A gene.

Cells transformed by proteins of early regions 1A (E1A) and 1B (E1B) of oncogenic adenovirus serotype 12 (Ad12) grow to tumours in syngeneic, immunocompetent rodents. To gain insight into the mechanisms of oncogenic transformation, we point mutated the first splice donor in the Ad12-E1A gene, leading to the loss of the Ad12-E1A(9.5S) and Ad12-E1A(11S/10S) proteins and to a conservative amino acid (aa) exchange at position aa 30 (valine vs. leucine) in the Ad12-E1A(13S) and Ad12-E1A(12S ) proteins. BMK cells transformed by mutant Ad12-E1A (Ad12-E1Am) plus Ad12-E1B via retrovirus-mediated gene transfer showed features comparable to wild-type Ad12-E1A (Ad12-E1Awt) plus Ad12-E1B-transformed cells: they formed foci in soft agar and produced tumours in immunodeficient nude mice, although after a prolonged latency period. These results suggest that Ad12-E1A(9.5S) and Ad12-E1A(11S/10S) are dispensable for cellular transformation. However, in contrast to Ad12-E1Awt cells, Ad12-E1Am cells failed to grow to tumours in syngeneic, immunocompetent rodents, with the exception of one cell line, which produced tumours in about 50% of the immunocompetent animals. Interestingly, the concentration of the putative tumour suppressor and co-activator p300 was elevated in cell lines expressing high levels of Ad12-E1A and Ad12-E1B due to an increased half-life. These results indicate that p300 is stabilized in Ad12-E1-transformed BMK cells, probably by a mechanism linked to high expression of Ad12-E1A/E1B.

Improved treatment of pancreatic cancer by IL-12 and B7.1 costimulation: antitumor efficacy and immunoregulation in a nonimmunogenic tumor model.

Ductal pancreatic adenocarcinoma is one of the commonest and most lethal cancers in the Western world. Unfortunately, recent advances in diagnostics, staging, and therapy in pancreatic carcinoma have not resulted in significant improvements in long-term survival. We have previously shown that adenovirus (Ad)-mediated coexpression of interleukin-12 (IL-12) and the costimulatory molecule B7.1 is extremely efficient in inducing regression of highly immunogenic transplanted and nontransplanted tumors. Here, we examined the antitumor efficacy of IL-12- and B7.1-based immunotherapy against a nonimmunogenic murine model of ductal pancreatic cancer. Compared with AdIL-12 treatment alone, single intratumoral injection of AdIL-12/B7.1 led to a prolonged immune response and mediated complete regression in 80% of treated animals. After rechallenge with parental tumor cells, 70% of cured mice remained tumor-free, suggesting that protective immunity had been induced. The antitumoral response was associated with upregulation of H-2K(b) and Abcb2 expression, whereas other components of the proteasome (Abcb3, Psmb9, and Psmb8) were not affected. These data indicate that upregulation of the antigen presentation machinery by AdIL-12/B7.1 may be a therapeutic rationale for nonimmunogenic, therapy-resistant pancreatic cancer.