Identification of a cancer stem cell-specific function for the histone deacetylases, HDAC1 and HDAC7, in breast and ovarian cancer.

Tumours are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumours in vivo. These cancer stem cells (CSCs) represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumour relapse. Despite this realization and great interest in CSCs, it has been difficult to develop CSC-targeted treatments due to our limited understanding of CSC biology. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the CSC phenotype. Utilizing a novel CSC model, we discovered that the HDACs, HDAC1 and HDAC7, are specifically over-expressed in CSCs when compared to non-stem-tumour-cells (nsTCs). Furthermore, we determine that HDAC1 and HDAC7 are necessary to maintain CSCs, and that over-expression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that clinically available HDAC inhibitors (HDACi) targeting HDAC1 and HDAC7 can be used to preferentially target CSCs. These results provide actionable insights that can be rapidly translated into CSC-specific therapies.

Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor metastasis via STAT3-mediated Twist1 upregulation.

p27 restrains normal cell growth, but PI3K-dependent C-terminal phosphorylation of p27 at threonine 157 (T157) and T198 promotes cancer cell invasion. Here, we describe an oncogenic feedforward loop in which p27pT157pT198 binds Janus kinase 2 (JAK2) promoting STAT3 (signal transducer and activator of transcription 3) recruitment and activation. STAT3 induces TWIST1 to drive a p27-dependent epithelial-mesenchymal transition (EMT) and further activates AKT contributing to acquisition and maintenance of metastatic potential. p27 knockdown in highly metastatic PI3K-activated cells reduces STAT3 binding to the TWIST1 promoter, TWIST1 promoter activity and TWIST1 expression, reverts EMT and impairs metastasis, whereas activated STAT3 rescues p27 knockdown. Cell cycle-defective phosphomimetic p27T157DT198D (p27CK-DD) activates STAT3 to induce a TWIST1-dependent EMT in human mammary epithelial cells and increases breast and bladder cancer invasion and metastasis. Data support a mechanism in which PI3K-deregulated p27 binds JAK2, to drive STAT3 activation and EMT through STAT3-mediated TWIST1 induction. Furthermore, STAT3, once activated, feeds forward to further activate AKT.

Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells.

Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation.

Tubal and ovarian pathways to pelvic epithelial cancer: a pathological perspective.

Prolongation of ovarian epithelial cancer survival depends on early detection or improved responses to chemotherapy. Gains in either have been modest at best. Understanding the diverse pathogenesis of this disease is critical to early intervention or prevention. This review addresses six important variables, including (i) cell of origin, (ii) site of origin, (iii) initial genotoxic events, (iv) risks imposed by hereditary and other promoting conditions, (v) subsequent factors that promote different patterns of metastatic spread, and (vi) prospects for intervention. This review proposes two distinct pathways to pelvic epithelial cancer. The first initiates in ovarian surface epithelium (OSE), Mullerian inclusions or endometriosis in the ovary. The second arises from the endosalpinx and encompasses a subset of serous carcinomas. The serous carcinogenic sequence in the distal fallopian tube is described and contrasted with lower grade serous tumors based on tumour location, earliest genetic change and ability (or lack of) to undergo terminal (ciliated) differentiation. Ultimately, a clear understanding of tumour origin and the mechanism(s) leading to the earliest phases of the serous and endometrioid carcinogenic sequences may hold the greatest promise for designing prevention strategies and/or developing new therapies.

Fatal necrotizing pneumonia caused by group A streptococcus.

Group A streptococcus (GAS) causes invasive, non-invasive and non-suppurative diseases. Pneumonia is one of the invasive infections caused by GAS. Although GAS is a significant and serious cause of childhood pneumonia, it is often overlooked clinically. Similarly, the recent literature is surprisingly scant on reports of GAS pneumonia and concentrates mainly on varicella-associated invasive GAS diseases. In this case report, we present a previously healthy 7-year-old child with community-acquired pneumonia that progressed rapidly and resulted in sepsis, respiratory failure and death. In both blood and pleural fluid cultures, Streptococcus pyogenes were isolated. On autopsy, macroscopic examination revealed that the lung tissue appeared to have lost its normal architecture. Necrosis was present and the lung had a spongy appearance with some solid areas. The light microscopy revealed massive oedema, haemorrhages, intense inflammatory cell infiltration and necrosis. This case report highlights the need for consideration of invasive GAS infection in the event of severe, rapidly progressing pneumonia in children.

Optimization of a versatile in vitro transcription assay for the expression of multiple start site TATA-less promoters.

Previous work from our laboratory has allowed for the subdivision of RNA polymerase II TATA-less promoters into two classes: those that initiate at a single start site (SSS) and those that initiate at multiple start sites (MSS). MSS promoters are defined by the lack of a TATA box and the presence of a transcription initiation window and a downstream MED-1 element (GCTCCC/G) [Ince, T. A., and Scotto, K. W. (1995) J. Biol. Chem. 270, 30249-30252]. Further insight into the mechanisms regulating TATA-less MSS promoters has been hampered by the lack of an in vitro transcription assay in which multiple start sites can be reproduced. In the present study, we describe the development of a versatile in vitro transcription system optimized for the expression of MSS promoters, termed the multiple promoter comparison (MPC) assay. By alteration of assay parameters including template length, cation and nucleotide concentrations, and RNA isolation method, the accurate and robust transcription of two MSS promoters, pgp1 (hamster P-glycoprotein class I homologue) and HPRT (human hypoxanthine phosphoribosyltransferase), was accomplished. Moreover, both TATA-containing and TATA-less single start site promoters were also transcribed in the MPC assay, making this the first general in vitro transcription system for the simultaneous analysis of all three classes of RNA polymerase II genes.

Molecular identification of latent precancers in histologically normal endometrium.

Discovery of somatically mutated cells in human tissues has been less frequent than would be predicted by in vitro mutational rates. We analyzed the PTEN tumor suppressor gene as an early marker for endometrial carcinogenesis, and we show that 43% of histologically normal premenopausal endometria contain rare glands that fail to express PTEN protein because of mutation and/or deletion. These persist between menstrual cycles. Histopathology of PTEN-null glands is initially unremarkable, but with progression, they form distinctive high-density clusters. These data are consistent with a progression model in which initial mutation is not rate limiting.

Transcriptional repression by p53 through direct binding to a novel DNA element.

The tumor suppressor protein p53 has been well documented as a transcriptional activator involved in the regulation of a number of critical genes involved in the cell cycle, response to DNA damage, and apoptosis. Activation by p53 requires the interaction of the protein with a consensus binding site consisting of two half-sites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head and separated by 0-13 base pairs. In addition to activation, p53 has been shown to be a potent repressor of transcription. However, the basis for p53-mediated repression is not well understood and has been proposed to occur indirectly through interactions with other promoter-bound transcription factors. In the present study, we show that p53 can repress transcription directly by binding to a novel head-to-tail (HT) site within the MDR1 promoter. A mutation that disrupted p53 binding to the MDR1 HT site blocked p53-mediated repression of the MDR1 promoter in transfection assays. Replacement of the HT site with a head-to-head (HH) site converted the activity of p53 from repression to activation, indicating that simple recruitment of p53 to the promoter is not sufficient for repression and that the orientation of the binding element determines the fate of p53-regulated promoters.

Surrogate biomarkers of HPV infection in cervical neoplasia screening and diagnosis.

The current prevention of cervical cancer and elimination of its precursors is predicated on the identification of cervical cytologic abnormalities and their histologic confirmation. This strategy, although effective, depends on both sensitivity and specificity of cytology and precise histologic distinction between precursor lesions and their mimics during biopsy interpretation. The effective application of diagnostic criteria is operator dependent and varies as a function of experience and training. However, because human papilloma viruses (HPV) are causative agents and alter the cell cycle in cervical neoplasms, host genes interacting directly or indirectly with HPV oncoproteins have been identified in vitro. Recent research has centered on identifying the host genes upregulated in association with HPV infection, determining their suitability as "surrogate markers" for HPV infection, and using these markers to identify HPV-associated epithelial lesions in tissue or cytologic specimens. This review surveys recent advances in this field, summarizing the advantages and limitations of several candidate biomarkers, including PCNA, Ki-67, cyclin E, p16ink4, MN antigen, carcinoembryonic antigen (CEA), and telomerase in the recognition of preinvasive cervical neoplasia, and discusses their future potential in cervical cancer screening. Based on current studies, the strongest candidates for diagnosis and screening are p16 and cyclin E (squamous) and MN (glandular) biomarkers. As new genes are identified and tested, the concept of biomarkers as tools in primary screening and lesion recognition will continue to mature.

Stable transfection of the P-glycoprotein promoter reproduces the endogenous overexpression phenotype: the role of MED-1.

Cellular resistance to multiple chemotherapeutic agents is most often due to the overexpression of P-glycoprotein (Pgp). The mechanisms(s) underlying Pgp overexpression had not been determined, due, in part, to a failure to reproduce the overexpression in transient transfection assays. We now report that stable transfection of a Pgp (pgp1) promoter/luciferase construct in the drug-sensitive Chinese hamster cell line DC-3F and its drug-resistant sublines reproduced the overexpression phenotype, with up to 18-fold higher activity observed in the resistant cell lines compared with DC-3F. Moreover, mutation of a pgp1 promoter element, multiple start site element downstream (MED-1), decreased transcription in drug-resistant cells without affecting activity in drug-sensitive cells. This is the first report of a Pgp promoter element differentially regulated in drug-resistant cells. Moreover, these data suggest that the regulation of Pgp transcription is modulated by chromatin structure, and that stable transfection may be more suitable for identifying promoter elements important for overexpression in drug-resistant cells.

A conserved downstream element defines a new class of RNA polymerase II promoters.

Although many TATA-less promoters transcribed by RNA polymerase II initiate transcription at multiple sites, the regulation of multiple start site utilization is not understood. Beginning with the prediction that multiple start site promoters may share regulatory features and using the P-glycoprotein promoter (which can utilize either a single or multiple transcription start site(s)) as a model, several promoters with analogous transcription windows were grouped and searched for the presence of a common DNA element. A downstream protein-binding sequence, MED-1 (Multiple start site Element Downstream), was found in the majority of promoters analyzed. Mutation of this element within the P-glycoprotein promoter reduced transcription by selectively decreasing utilization of downstream start sites. We propose that a new class of RNA polymerase II promoters, those that can utilize a distinctive window of multiple start sites, is defined by the presence of a downstream MED-1 element.

Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells.

The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated. We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene (Pgp1) in several MDR Chinese hamster cell lines. The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CHRC5, express a level of Pgp1 RNA commensurate with the increase in Pgp1 dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgp1 mRNA without a concomitant increase in Pgp1 gene-copy number. Analysis of Pgp1 transcription start point (tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CHRC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines 'switch' to a more complex pattern, using four additional Pgp1 tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+1). This observation of a difference in the regulation of transcription of Pgp in MDR vs. drug-sensitive cells suggests that the 'switch' in tsp selection may be involved in the increased expression of Pgp1 mRNA. Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype. Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.

Fibroblast matrix and surface components that mediate cell-to-cell interaction with lymphocytes.

The interaction between lymphocytes and fibroblasts in vitro has been examined using a quantitative ELISA assay to measure the binding of T and B cells to monolayer cultures of human dermal fibroblasts. This was carried out on microtiter culture plates, using an anti-Thy-1 monoclonal antibody, to determine the attachment of murine T lymphocytes and an affinity-purified polyclonal anti-IgM antibody to measure B cell binding. Both types of lymphocyte were found to adhere strongly to intact human fibroblasts, and also had high levels of attachment to purified fibroblast plasma membranes and extracts of the fibroblast extracellular matrix. Attachment, particularly of B lymphocytes, also took place onto plastic surfaces coated with fibronectin, but not to collagens or to intact fibroblasts that had been fixed with a low concentration of paraformaldehyde. Lymphocyte binding to fibroblasts was partially prevented by a monoclonal antibody against fibroblast MHC class II antigens, but not against the class I membrane complex, or by polyclonal antiserum to the cell surface mannose 6-phosphate receptor. In addition, although both lymphocyte types were able to adhere to fibro-nectin, the presence of antibody against fibronectin or the synthetic peptide Arg-Gly-Asp-Ser, had no effect on their attachment to fibroblasts. Thus, lymphocyte adhesion may occur by fibronectin, but other types of interactions with fibroblasts also appear to take place.