No abstract

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells.

The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.

Purpose Morphologically heterogeneous prostate cancers (PC) that behave clinically like small cell PC (SCPC) share their chemotherapy responsiveness. We asked whether these clinically defined, morphologically diverse, ‘aggressive variant PC’ (AVPC) also share molecular features with SCPC. Experimental Design 59 PC samples from 40 clinical trial participants meeting AVPC criteria, and 8 patient-tumor derived xenografts (PDX) from 6 of them, were stained for markers aberrantly expressed in SCPC. DNA from 36 and 8 PDX was analyzed by Oncoscan® for copy number gains (CNG) and losses (CNL). We used the AVPC PDX to expand observations and referenced publicly available data sets to arrive at a candidate molecular signature for the AVPC. Results Irrespective of morphology, Ki67 and Tp53 stained ≥ 10% cells in 80% and 41% of samples respectively. RB1 stained <10% cells in 61% of samples and AR in 36%. MYC (surrogate for 8q) CNG and RB1 CNL showed in 54% of 44 samples each and PTEN CNL in 48%. All but 1 of 8 PDX bore Tp53 missense mutations. RB1 CNL was the strongest discriminator between unselected castration resistant PC (CRPC) and the AVPC. Combined alterations in RB1, Tp53 and/or PTEN were more frequent in the AVPC than in unselected CRPC and in The Cancer Genome Atlas samples. Conclusions Clinically defined AVPC share molecular features with SCPC and are characterized by combined alterations in RB1, Tp53 and/or PTEN.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the alpha 2(I) collagen, the alpha 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the pro␣1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3 repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465-and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.Acquisition of the chondrocyte phenotype by mesenchymal cells is one of the major pathways of differentiation of these cells. Chondrocytes form several types of cartilages including the growth plate cartilages essential to skeletal formation and cartilages that have supporting roles and persist throughout adult life such as the articular cartilages and the cartilages of the nose, ear, and trachea. Chondrocyte differentiation presumably involves first the commitment of undifferentiated mesenchymal cells to the chondrocyte lineage (1). Cell condensation and further maturation lead to a fully differentiated phenotype characterized by the synthesis of cartilage extracellular matrix proteins, including collagen types II, IX, and XI, the large proteoglycan aggrecan, the link protein, and the cartilage oligomeric protein (24). Recent molecular and biochemical studies with cell culture, gene inactivation experiments with mice, and the identification of genes responsible for mouse and human skeletal abnormalities have documented the importance of growth and differentiation factors, extracellular matrix proteins, signaling mediators, and tr...

macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.The major histocompatibility complex (MHC) class II proteins play a central role in the immune response. Extensive analysis has underscored that much of the fluctuation in class II MHC antigen expression can be attributed to changes at the transcriptional level (46,47). In addition to the class II MHC molecules themselves, associative accessory molecules that are necessary for class II antigen MHC function appear to be controlled in a similar fashion. These associative molecules include the MHC class II-associated invariant chain (Ii) and the more recently described DM heterodimer. All class II MHC, Ii, and DM promoters share the unique presence of three DNA elements, called W, X, and Y, which are highly conserved and critical for promoter function (2, 15). The W-X-Y elements are not only important for constitutive gene expression in B cells but also critical for inducible gene expression. In addition to the conservation in sequence, the spacing between the X and Y elements is highly conserved at approximately two helical turns. Increasing the number of helical turns between these two elements preserves function, while disrupting this orientation destroys promoter activation. Our group previously hypothesized that this restrictive spacing may be required to align the X and Y elements on the same side of the DNA helix, thus allowing transcription factors which can bind these elements to directly interact or to participate in the assembly of a larger promoter complex (48,49).The Y box is a CCAAT motif, and it interacts with NF-Y/ CBF (also known as YEBP/CP-1). NF-Y/CBF is composed of A, B, and C subunits (26,27,57), with the conserved core sequences of NF-YC (CBF-C) and NF-YB (CBF-A) forming a histone fold motif similar to the nucleosome subunits H2A and H2B (1). NF-Y/CBF plays a critical role in opening chromatin because mutation of the NF-Y/CBF-binding sites in both the DRA and Ii promoters results in the loss of protein binding across these promoters in intact cells (24,54). NF-Y/CBF can preset chromatin for other transcriptional coactivators, such as the histone acetylase GCN5, p300, and pCAF (10,19,23). The X box is a bipartite sequence. X1 is bound by the trimeric transcription factor, RFX, formed by RFX5, RFXAP, and RFXANK/RFXB (12,28,45). The lack of RFX results in several subclasses of bare lymphocyte syndrome (BLS), a severe immunodeficiency attributed to the lack of class II MHC expression. RFX is required for both the constitutive and gamma interferon (IFN-␥) induction of class II MHC expression (5). The X2 element binds a protein complex, X2BP, which has been recently identified as the CREB protein (29).Despite the extensive demonstration that X and Y boxbinding proteins are important for class II MHC regulation, these proteins are constitutively expressed and cannot explain the cell-, tissue-, developmentally, and cytokine-inducible expression of class ...