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Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n=3 mice per group; 5% vs 46%, difference=41%, 95% confidence interval=15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1-epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers. DOI: 10.1093/jnci/djp458

Background: Dominant-activating mutations in the RET protooncogene, a receptor tyrosine kinase, have been identified as a cause of medullary thyroid carcinoma. Such oncogenic RET mutations induce its ligand-independent constitutive trans-autophosphorylation. We investigated the role of endogenous oncogenic RET autophosphorylation in maintaining the neoplastic phenotype in medullary thyroid carcinoma cells and orthotopic medullary thyroid carcinomas in RET transgenic mice. Methods: We constructed adenoviral vectors expressing a dominant-negative truncated form of RET, termed RE[T.sup.[DELTA]TK], and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RE[T.sup.[DELTA]TK] on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RE[T.sup.[DELTA]TK] in primary medullary thyroid carcinomas in transgenic mice was assessed by monitoring tumor growth. All statistical tests were two-sided. Results: Cell viability was reduced. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK), components of downstream signal transduction pathways, was abolished, and cell cycle progression was reduced. Expression of cell cycle regulator cyclin D1 was decreased, and expression of cell cyle regulators [p21.sup.CIP1/WAF1] and [p27.sup.KIP1] was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RE[T.sup.[DELTA]TK] with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RE[T.sup.[DELTA]TK] into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RE[T.sup.[DELTA]TK] (43.6%, 95% confidence interval [CI] = 30.7% to 56.5% ; P = .010; two-sided unpaired Student's t test), whereas tumors in mice treated with a control vector were larger than their initial size (139.8%, 95% CI = 120.3% to 159.3%; P

Background: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. Methods: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x [10.sup.9] plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. Results: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P = .006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of [CD4.sup.+] and [CD8.sup.+] T cells and interferon gamma (IFN [Gamma]). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of [CD4.sup.+] and [CD8.sup.+] T cells and IFN [Gamma]) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated ([is greater than or equal to] 95%) 7 weeks after treatment. Conclusion: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans. [J Natl Cancer Inst 2001;93:472-9]