Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelialto-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imagingbased experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.carcinoma | receptor tyrosine kinase | breast cancer

In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIA␣␤ gene and posttranslationally cleaved into ␣ and ␤ subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1 ؊/؊ MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.In eukaryotes, initiation of RNA polymerase II transcription requires the assembly of a preinitiation complex. Specific binding of TBP to promoters is a key step in the formation of PIC, which is followed by recruitment of general transcription factors and polymerase II. The basal transcription factor TFIIA interacts with TBP and stabilizes its binding to DNA (26,28). TFIIA has also been shown to interact with several activators (11,12,18,27) and is required for transcriptional activation of certain genes (10,13,14,20,21).In higher eukaryotes, purified TFIIA is composed of three subunits, ␣, ␤, and ␥. TFIIA␣␤ is encoded by a single gene and cleaved posttranslationally into ␣ and ␤ subunits. The ␥ subunit is conserved among different species, whereas sequence similarity in TFIIA␣␤ is limited mostly to the N-terminal region of the ␣ subunit and the C terminus covering most of the ␤ subunit (19). Recently, the cleavage recognition site (CRS) that is essential for TFIIA cleavage has been identified as QVDG (amino acids [aa] 272 to 275), and the N terminus of the ␤ subunit was determined to be at D278, located 3 amino acids downstream of the CRS (Fig. 1B) (6). The CRS is remarkably similar in different evolutionarily distinct species and is embedded in an otherwise nonconserved and probably unstructured region (1, 4, 24). The germ cell-specific TFIIA-like factor ALF, a TFIIA variant that contains the CRS, was also shown to be cleaved (5, 6). TFIIA cleavage was first reported more than a decade ago (26), and it has been generally assumed that uncleaved TFIIA is the precursor and cleavage occurs to activate TFIIA for transcription. Both uncleaved ␣␤ and the cleaved ␣ and ␤ subunits can be found in association with the TFIIA␥ subunit in vivo (15, 16), and both forms interact with TBP on DNA and support transcription to similar extents in vitro and in reporter assays (6,22). TFIIA is mainly found in the cleaved form (␣ plus ␤ plu...

BackgroundFor safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations.ResultsWe see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set.ConclusionsHistone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

The transcription factor TFIIA is encoded by two genes, TFIIAab and TFIIAc. In higher eukaryotes, the TFIIAab is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAb subunit starts at Asp 278. We found that a cleavage recognition site (CRS), a string of amino acids QVDG at positions À6 to À3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAb greatly affect degradation of the b-subunit. We propose that cleavage and subsequent degradation fine-tune the amount of TFIIA in the cell and consequently the level of transcription.

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The transcription factor SOX9 is believed to be the master regulator of chondrogenesis. SOX8 is another SOX group E transcription factor with a high degree of homology to SOX9. Here, we demonstrate that SOX8 mRNA levels decrease during in vitro dedifferentiation of human articular chondrocytes and increase during chondrogenic differentiation of mesenchymal stromal cells. Knockdown of SOX9 reduced the expression of SOX8, COL2A1, and a range of other chondrogenic molecules. SOX8 knockdown reduced the expression of a large number of overlapping chondrogenic molecules, but not SOX9. Neither siSOX9 nor siSOX8 altered expression of the hypertrophic marker gene COL10A1. siSOX9, but not siSOX8 led to upregulation of hypertrophy associated genes MMP13 and ALPL. Transfection of synthetic SOX5, 6, and 9 mRNA trio upregulated SOX8, COL2A1, and ACAN, but not COL10A1 mRNA. Replacement of synthetic SOX9 by SOX8 in the SOX trio showed similar but lower chondrogenic effect. We conclude that SOX8 expression is regulated by SOX9, and that both together with SOX5 and SOX6 are required as a SOX quartet for transcription of COL2A1 and a large number of other chondrogenic molecules. Neither SOX8 nor SOX9 affect COL10A1 expression, but SOX9 inhibits chondrocyte hypertrophy through inhibition of MMP13 and ALPL expression.

Metastasis underlies the majority of cancer-related deaths. Hence, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMT) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in mammography-detected primary breast cancers independently predicts strongly reduced overall patient survival, and matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by epithelial-to-mesenchymal transition in pre-malignant mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Using epi-allelic RNA interference analysis in metastatic breast cancer cells we delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness, and to form tumors in foreign and tissue engineered microenvironments in vivo. Importantly, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs, and increased overall survival, in two different optical imaging-based experimental breast cancer models. Thus, Axl represents a novel downstream effector of tumor cell EMT that is required for breast cancer metastasis. The detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B105.

BackgroundThe dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.ResultsIn order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.ConclusionThe unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.