A cyclic peptide inhibitor of HIF-1 heterodimerization that inhibits hypoxia signaling in cancer cells.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1ß protein-protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein-protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

MicroRNAs: critical regulators of epithelial to mesenchymal (EMT) and mesenchymal to epithelial transition (MET) in cancer progression.

MicroRNAs (miRNAs) are a class of small highly conserved RNAs that provide widespread expressional control through the translational repression of mRNA. MiRNAs have fundamental roles in the regulation of intracellular processes, and their importance during malignant transformation and metastasis is becoming increasingly well recognized. An important event in the metastatic cascade is epithelial to mesenchymal transition (EMT), a reversible phenotypic switch over, which endows malignant epithelial cells with the capacity to break free from one another and invade the surrounding stroma. Our understanding of EMT has been significantly improved by the characterization of miRNAs that influence the signalling pathways and downstream events that define EMT on a molecular level. Here, we detail the role of miRNAs in EMT, and in doing so demonstrate their importance in the early stages of the metastatic cascade; we discuss a significant body of data that suggest new opportunities for drug development, and we highlight critical knowledge gaps that remain to be addressed.

Bcl-2-associated athanogene-1 (BAG-1): a transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification.

BAG-1, an anti-apoptotic protein, was identified by its ability to bind to BCL-2, HSP70-family molecular chaperones and nuclear hormone receptor family members. Two BAG-1 isoforms, BAG-1L (50 kDa) and BAG-1S (32 kDa) were identified in mouse cells and BAG-1 expression was reported in murine growth plate and articular chondrocytes. The present study aimed to elucidate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification. In long bones of skeletally immature mice, we observed expression of BAG-1 in the perichondrium, osteoblasts, osteocytes in the bone shaft, bone marrow, growth plate and articular chondrocytes. Monolayer cultures of murine chondrocytic ATDC5 cells, which exhibited robust expression of both BAG-1 isoforms and the Bag-1 transcript, were utilized as an in vitro model to delineate the roles of BAG-1. Overexpression of BAG-1L in ATDC5 cells resulted in downregulation of Col2a1 expression, a gene characteristically downregulated at the onset of hypertrophy, and an increase in transcription of Runx-2 and Alkaline phosphatase, genes normally expressed at the onset of chondrocyte hypertrophy and cartilage mineralization in the process of endochondral ossification. We also demonstrated the anti-apoptotic role of BAG-1 in chondrocytes as overexpression of BAG-1 protected ATDC5 cells, which were subjected to heat-shock at 48 degrees C for 30 min, against heat-shock-induced apoptosis. Overexpression of the SOX-9 protein in ATDC5 cells resulted in increased Bag-1 gene expression. To further investigate the regulation of Bag-1 gene expression by SOX-9, CHO cells were co-transfected with the human Bag-1 gene promoter-Luciferase reporter construct and the human pSox-9 expression vector. Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein. In conclusion, a novel finding of this study is the role of BAG-1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX-9 and the anti-apoptotic role of BAG-1 in chondrocytic cells. Modulation of Bag-1 expression can therefore mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification.

Stratifying risk of recurrence in stage II colorectal cancer using deregulated stromal and epithelial microRNAs.

MicroRNAs (miRNAs) enable colonic epithelial cells to acquire malignant characteristics and metastatic capabilities. Recently, cancer relevant miRNAs deregulated during disease progression have also been identified in tumor-associated stroma.By combining laser-microdissection (LMD) with high-throughput screening and high-sensitivity quantitation techniques, miRNA expression in colorectal cancer (CRC) specimens and paired normal colonic tissue was independently characterized in stromal and epithelial tissue compartments. Notably, deregulation of the key oncogene miR-21 was identified exclusively as a stromal phenomenon and miR-106a, an epithelial phenomenon in the malignant state.MiRNAs identified in this study successfully distinguished CRC from normal tissue and metastatic from non-metastatic tumor specimens. Furthermore, in a separate cohort of 50 consecutive patients with CRC, stromal miR-21 and miR-556 and epithelial miR-106a expression predicted short disease free survival (DFS) and overall survival (OS) in stage II disease: miR-21 (DFS: HR = 2.68, p = 0.015; OS: HR = 2.47, p = 0.029); miR-556 (DFS: HR = 2.60, p = 0.018); miR-106a (DFS: HR = 2.91, p = 0.008; OS: HR = 2.25, p = 0.049); combined (All High vs. All Low. DFS: HR = 5.83, p = 0.002; OS: HR = 4.13, p = 0.007).These data support the notion that stromal as well as epithelial miRNAs play important roles during disease progression, and that mapping patterns of deregulated gene expression to the appropriate tumor strata may be a valuable aid to therapeutic decision making in CRC.

Molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts.

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade. Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques. Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions. Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision. By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.

Induction of cytosolic calcium flux by CD20 is dependent upon B Cell antigen receptor signaling.

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.