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Purpose: DEAD box protein 3 (DDX3) is an RNA helicase with oncogenic properties that shuttles between the cytoplasm and nucleus. The majority of DDX3 is found in the cytoplasm, but a subset of tumors has distinct nuclear DDX3 localization of yet unknown biological significance. This study aimed to evaluate the significance of and mechanisms behind nuclear DDX3 expression in colorectal and breast cancer. Methods: Expression of nuclear DDX3 and the nuclear exporter chromosome region maintenance 1 (CRM1) was evaluated by immunohistochemistry in 304 colorectal and 292 breast cancer patient samples. Correlations between the subcellular localization of DDX3 and CRM1 and the difference in overall survival between patients with and without nuclear DDX3 were studied. In addition, DDX3 mutants were created for in vitro evaluation of the mechanism behind nuclear retention of DDX3. Results: DDX3 was present in the nucleus of 35% of colorectal and 48% of breast cancer patient samples and was particularly strong in the nucleolus. Nuclear DDX3 correlated with worse overall survival in both colorectal (hazard ratio [HR] 2.34, P<0.001) and breast cancer (HR 2.39, P=0.004) patients. Colorectal cancers with nuclear DDX3 expression more often had cytoplasmic expression of the nuclear exporter CRM1 (relative risk 1.67, P=0.04). In vitro analysis of DDX3 deletion mutants demonstrated that CRM1-mediated export was most dependent on the N-terminal nuclear export signal. Conclusion: Overall, we conclude that nuclear DDX3 is partially CRM1-mediated and predicts worse survival in colorectal and breast cancer patients, putting it forward as a target for therapeutic intervention with DDX3 inhibitors under development in these cancer types. [ABSTRACT FROM AUTHOR]

Background: The basic helix-loop-helix transcription factor TWIST1 (Twist) is involved in embryonic cell lineage determination and mesodermal differentiation. There is evidence to indicate that Twist expression plays a role in breast tumor formation and metastasis, but the role of Twist in dysregulating pathways that drive the metastatic cascade is unclear. Moreover, many of the genes and pathways dysregulated by Twist in cell lines and mouse models have not been validated against data obtained from larger, independant datasets of breast cancer patients.Methods: We over-expressed the human Twist gene in non-metastatic MCF-7 breast cancer cells to generate the estrogen-independent metastatic breast cancer cell line MCF-7/Twist. These cells were inoculated in the mammary fat pad of female severe compromised immunodeficient mice, which subsequently formed xenograft tumors that metastasized to the lungs. Microarray data was collected from both in vitro (MCF-7 and MCF-7/Twist cell lines) and in vivo (primary tumors and lung metastases) models of Twist expression. Our data was compared to several gene datasets of various subtypes, classes, and grades of human breast cancers.Results: Our data establishes a Twist over-expressing mouse model of breast cancer, which metastasizes to the lung and replicates some of the ontogeny of human breast cancer progression. Gene profiling data, following Twist expression, exhibited novel metastasis driver genes as well as cellular maintenance genes that were synonymous with the metastatic process. We demonstrated that the genes and pathways altered in the transgenic cell line and metastatic animal models parallel many of the dysregulated gene pathways observed in human breast cancers.Conclusions: Analogous gene expression patterns were observed in both in vitro and in vivo Twist preclinical models of breast cancer metastasis and breast cancer patient datasets supporting the functional role of Twist in promoting breast cancer metastasis. The data suggests that genetic dysregulation of Twist at the cellular level drives alterations in gene pathways in the Twist metastatic mouse model which are comparable to changes seen in human breast cancers. Lastly, we have identified novel genes and pathways that could be further investigated as targets for drugs to treat metastatic breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: Twist is a basic helix loop helix protein that plays a role both in human development and in cancer biogenesis. While characterizing the effects of Twist on breast epithelial cell transformation, we identified E-cadherin as a target gene that is down-regulated by Twist. In this study, we demonstrate that Twist can transcriptionally repress E-cadherin in breast cancer cells. Using transient promoter assays, we show that Twist can down-regulate E-cadherin promoter activity by up to two folds. This is further supported by immunoblot analyses which indicates that over-expression of Twist decreases E-cadherin protein levels in breast cancer cell lines. Subsequently, chromatin immunoprecipitation performed on MCF-7/Twist and Hs578 T (high level of endogenous Twist expression) confirmed Twist binding to the E-cadherin promoter. Finally, the functional relevance of this regulation was verified by quantitative real-time PCR and immunohistochemistry on a cohort of breast cancer samples. [Copyright &y& Elsevier]