Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Protein–protein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes, Med7N/21, Med7C/31 and Med4/9. The subunits, Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4.

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MicroRNAs (miRNAs) are short single-stranded RNA molecules that have a critical role in the regulation of gene expression. Alterations in miRNA expression levels have been observed in multiple tumor types and there is clear evidence on their active involvement in cancer development. Here, a comprehensive miRNA expression profiling in 16 pancreatic cancer cell lines and four normal pancreatic samples provided a specific molecular signature for pancreatic cancer and enabled us to identify 72 differentially expressed miRNAs with approximately half of them being up- and half downregulated in cancer cells as compared with normal samples. Of these, miR-31 was selected for further functional analyses based on its interesting "on-off" type expression profile, i.e., very low or even absent expression in normal pancreas and in six of the pancreatic cancer samples but extremely high expression in the remaining 10 cell lines. Quite unexpectedly, both the inhibition of miR-31 in AsPC-1 and HPAF-II pancreatic cancer cells with high endogenous expression and forced expression of miR-31 in MIA PaCa-2 with low endogenous levels led to reduced cell proliferation, migration, and invasion. More importantly, in AsPC-1 cells further enhancement of miR-31 also resulted in reduced cell migration and invasion, implicating that the level of miR-31 is critical for these phenotypes. This study highlights a specific miRNA expression pattern in pancreatic cancer and reveals that manipulation of miR-31 expression leads to reduced cell migration and invasion in pancreatic cancer.

Mediator complex subunit 29 (MED29) is part of a large multiprotein coactivator complex that mediates regulatory signals from gene-specific activators to general transcription machinery in RNA polymerase II mediated transcription. We previously found that MED29 is amplified and overexpressed in pancreatic cancer and that MED29 silencing leads to decreased cell survival in PANC-1 pancreatic cancer cells with high MED29 expression. Here we further demonstrate decreased migration, invasion and colony formation in PANC-1 cells after MED29 silencing. Unexpectedly, lentiviral-based overexpression of MED29 led to decreased proliferation of NIH/3T3 cells as well as MIAPaCa-2 pancreatic cancer cells with low endogenous expression. More importantly, subcutaneous inoculation of the MED29-transduced pancreatic cancer cells into immuno-compromised mice resulted in dramatic tumor suppression. The mock-control mice developed large tumors, whereas the animals with MED29-xenografts showed both decreased tumor incidence and a major reduction in tumor size. Gene expression analysis in the MED29-transduced pancreatic cancer cells revealed differential expression of genes involved in control of cell cycle and cell division. The observed gene expression changes are expected to modulate the cell cycle in a way that leads to reduced cell growth, explaining the in vivo tumor suppressive phenotype. Taken together, these data implicate MED29 as an important regulator of key cellular functions in pancreatic cancer with both oncogenic and tumor suppressive characteristics. Such a dualistic role appears to be more common than previously thought and is likely to depend on the genetic background of the cancer cells and their surrounding environment.

MicroRNAs (miRNAs) are short single-stranded RNA molecules which have a critical role in the regulation of gene expression. Several miRNAs have been shown to be up- or downregulated in different tumor types and there is clear evidence that they are actively involved in cancer development. Depending on their target genes, microRNAs can act both as oncogenes or tumor suppressor genes. Identification of differentially expressed miRNAs and their target genes in cancer is likely to provide essential information on disease pathogenesis and thereby new tools for the clinical management of cancer. Here we aimed to identify differentially expressed miRNAs in pancreatic cancer and to explore their possible role in pancreatic cancer pathogenesis. To this end, we screened miRNA expression levels in 16 established pancreatic cancer cell lines and four normal human pancreatic RNA samples using miRNA arrays. Hierarchical clustering of the miRNA expression data separated the normal and cancer samples into two distinct clusters, indicating a specific miRNA signature in pancreatic cancer different from that seen in normal pancreas. The data analysis (eBayes method and the Benjamini-Hochberg adjustment) revealed a subset of 72 miRNAs which were either up- or downregulated (at least 1.5-fold, adjusted p-value <0.05) in pancreatic cancer compared with the normal samples. Among these were miRNAs previously linked to cancer, for example miR-21 and members of the let-7 family, but also novel differentially expressed miRNAs were identified. Twelve of the differentially expressed miRNAs were validated by qRT-PCR and a median Pearson correlation value of 0.83 was observed between the two methods, thus confirming the reliability of the microarray results. Of the 72 differentially expressed miRNAs, 35 had increased and 37 decreased expression in cancer samples. This is an interesting finding since miRNAs are generally believed to be more often downregulated than upregulated in cancer. Evaluation of the genomic localization of the differentially expressed miRNAs showed clustering at a few specific chromosomal sites suggesting that genomic aberrations might partly underlie the observed expression changes. To identify possible target genes for the differentially expressed miRNAs, we used the GOmir application which utilizes four different target prediction programs. A range of targets were identified, some of which were common for all four programs and thus represent the most probable target genes. Finally, selected miRNAs showing the most frequent differential expression in pancreatic cancer will be characterized and the functional consequences of their aberrant expression on cancer cell phenotype will be studied. By understanding the connections between miRNA expression, gene expression and cancer cell characteristics, we may be able to find new targets for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3043.

Bone morphogenetic proteins (BMP) are signaling molecules involved in diverse developmental phases and are able to regulate cell growth, apoptosis and differentiation. For the last decade, they have also been increasingly in focus in cancer research. Studies have demonstrated that BMPs are able to both promote and inhibit cancer progression. In pancreatic cancer, there are only very few papers that have evaluated the role of BMP signaling. In this work, we have first determined the expression of seven BMP ligands (BMP2- BMP8) and six BMP specific receptors in 16 pancreatic cancer cell lines and in 4 normal pancreatic tissues using qRT-PCR. Expression of six BMP specific receptors was seen in all pancreatic cancer cell lines as well as in normal pancreas samples indicating that these cells are able to respond to BMP signals. Transcripts of the ligands BMP2, BMP3, BMP6 and BMP7 were detected at similar expression levels in both cancer and normal sample groups. The relative expression levels of BMP5 and BMP8 were significantly decreased in cancer cell lines compared to the normal pancreas samples. Interestingly, in a subset (4/16) of the cancer cell lines BMP4 was detected at highly elevated expression level whereas in rest of the cancer and in all normal samples its expression was barely detectable. We chose to study further the potential role of BMP4 and BMP5 signaling in pancreatic cancer pathogenesis. Changes in cell growth, migration, and invasion were examined in five pancreatic cancer cell lines (AsPC-1, SU.86.86, MIAPaCa-2, HPAF-II, and PANC-1) after recombinant BMP4 or BMP5 treatment. BMP5 treatment reduced cell number in three cell lines up to 36% (MIAPaCa-2, HPAF-II, and PANC-1). BMP4 decreased growth in four cell lines with the most distinct difference seen in MIAPaCa-2 and PANC-1 cells (30% or 79% decrease in cell number, respectively). BMP5 and BMP4 induced growth inhibition were both mainly due the alterations in cell cycle. Despite the growth inhibitory effect, BMP5 simultaneously increased the migration and invasion of two cell lines (MIAPaCa-2 and HPAF-II). Similarly, BMP4 increased migration and invasion of three cell lines with clear growth reduction. BMP4 treatment resulted in 3.5-fold increase of invaded MIAPaCa-2 cells and 10.8-fold increase of invaded PANC-1 cells. All cell lines with phenotypic changes showed activation of the canonical SMAD pathway. In addition, phosphorylation of MAP-kinases p38 and ERK1/2 was detected in MIAPaCa-2 cells after BMP4 and BMP5 stimulation. Taken together, BMP4 and BMP5 are able to both inhibit the growth and promote migration and invasion of the same pancreatic cell line. In this regard they resemble a closely related cytokine, transforming growth factor β, with a demonstrated dual function in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4099.