Estrogen distinctly regulates transcription and translation of lncRNAs and pseudogenes in breast cancer cells.

Estrogen drives key transcriptional changes in breast cancer and stimulates breast cancer cells' growth with multiple mechanisms to coordinate transcription and translation. In addition to protein-coding transcripts, estrogen can regulate long non-coding RNA (lncRNA) transcripts, plus diverse non-coding RNAs including antisense, enhancer, and intergenic. LncRNA genes comprise the majority of human genes. The accidental, or regulated, translation of their short open reading frames by ribosomes remains a controversial topic. Here we report for the first time an integrated analysis of RNA abundance and ribosome occupancy level, using Ribo-seq combined with RNA-Seq, in the estrogen-responsive, estrogen receptor α positive, human breast cancer cell model MCF7, before and after hormone treatment. Translational profiling can determine, in an unbiased manner, which fraction of the genome is actually translated into proteins, as well as resolving whether transcription and translation respond concurrently, or differentially, to estrogen treatment. Our data showed specific transcripts more robustly detected in RNA-Seq than in the ribosome-profiling data, and vice versa, suggesting distinct gene-specific estrogen responses at the transcriptional and the translational level, respectively. Here, we showed that estrogen stimulation affects the expression levels of numerous lncRNAs, but not their association with ribosomes, and that most lncRNAs are not ribosome-bound. For the first time, we also demonstrated the transcriptional and translational response of expressed pseudogenes to estrogen, pointing to new perspectives for drug-target development in breast cancer in the future.

The Growth-Arrest-Specific (GAS)-5 Long Non-Coding RNA: A Fascinating lncRNA Widely Expressed in Cancers.

Long non-coding RNA (lncRNA) genes encode non-messenger RNAs that lack open reading frames (ORFs) longer than 300 nucleotides, lack evolutionary conservation in their shorter ORFs, and do not belong to any classical non-coding RNA category. LncRNA genes equal, or exceed in number, protein-coding genes in mammalian genomes. Most mammalian genomes harbor ~20,000 protein-coding genes that give rise to conventional messenger RNA (mRNA) transcripts. These coding genes exhibit sweeping evolutionary conservation in their ORFs. LncRNAs function via different mechanisms, including but not limited to: (1) serving as "enhancer" RNAs regulating nearby coding genes in cis; (2) functioning as scaffolds to create ribonucleoprotein (RNP) complexes; (3) serving as sponges for microRNAs; (4) acting as ribo-mimics of consensus transcription factors binding sites in genomic DNA; (5) hybridizing to other nucleic acids (mRNAs and genomic DNA); and, rarely, (6) as templates encoding small open reading frames (smORFs) that may encode short proteins. Any given lncRNA may have more than one of these functions. This review focuses on one fascinating case-the growth-arrest-specific (GAS)-5 gene, encoding a complicated repertoire of alternatively-spliced lncRNA isoforms. GAS5 is also a host gene of numerous small nucleolar (sno) RNAs, which are processed from its introns. Publications about this lncRNA date back over three decades, covering its role in cell proliferation, cell differentiation, and cancer. The GAS5 story has drawn in contributions from prominent molecular geneticists who attempted to define its tumor suppressor function in mechanistic terms. The evidence suggests that rodent Gas5 and human GAS5 functions may be different, despite the conserved multi-exonic architecture featuring intronic snoRNAs, and positional conservation on syntenic chromosomal regions indicating that the rodent Gas5 gene is the true ortholog of the GAS5 gene in man and other apes. There is no single answer to the molecular mechanism of GAS5 action. Our goal here is to summarize competing, not mutually exclusive, mechanistic explanations of GAS5 function that have compelling experimental support.

Evaluating a Decision Aid for Improving Decision Making in Patients with Early-stage Breast Cancer.

BACKGROUND: Early-stage breast cancer patients face a series of complex treatment decisions, with the first typically being choice of locoregional treatment. There is a need for tools to support patients in this decision-making process. METHODS: We developed an innovative, online locoregional treatment tool based on International Patient Decision Aids Standards criteria. We evaluated its impact on patient knowledge about treatment and appraisal of decision making in a pilot study using a clinical sample of newly diagnosed, breast cancer patients who were randomized to view the decision aid website first or complete a survey prior to viewing the decision aid. Differences in knowledge and decision appraisal between the two groups were compared using t-tests and chi-square tests. Computer-generated preferences for treatment were compared with patients' stated preferences using chi-square tests. RESULTS: One hundred and one newly diagnosed patients were randomized to view the website first or take a survey first. Women who viewed the website first had slightly higher, though not significantly, knowledge about surgery (p = 0.29) and reconstruction (p = 0.10) than the survey-first group. Those who viewed the website first also appraised their decision process significantly more favorably than did those who took the survey first (p < 0.05 for most decision outcomes). There was very good concordance between computer-suggested and stated treatment preferences. CONCLUSION: This pilot study suggests that an interactive decision tool shows promise for supporting early-stage breast cancer patients with complicated treatment decision making.

CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells.

Increased expression of lymphangiogenesis factors VEGF-C/D and heparanase has been correlated with the invasion of cancer. Furthermore, chemokines may modify matrix to facilitate metastasis, and they are associated with VEGF-C and heparanase. The chemokine CXCL7 binds heparin and the G-protein-linked receptor CXCR2. We investigated the effect of CXCR2 blockade on the expression of VEGF-C/D, heparanase, and on invasion. CXCL7 siRNA and a specific antagonist of CXCR2 (SB225002) were used to treat CXCL7 stably transfected MCF10AT cells. Matrigel invasion assays were performed. VEGF-C/D expression and secretion were determined by real-time PCR and ELISA assay, and heparanase activity was quantified by ELISA. SB225002 blocked VEGF-C/D expression and secretion (P < .01). CXCL7 siRNA knockdown decreased heparanase (P < .01). Both SB225002 and CXCL7 siRNA reduced the Matrigel invasion (P < .01). The MAP kinase signaling pathway was not involved. The CXCL7/CXCR2 axis is important for cell invasion and the expression of VEGF-C/D and heparanase, all linked to invasion.

Immunohistochemical analysis of ocular platelet basic protein expression during infection with Pseudomonas aeruginosa.

Platelet basic protein (PBP) and several of its derivatives are known to express a wide range of biological characteristics. It is the precursor of connective tissue activating peptide (CTAP-III), beta thromboglobulin (beta-TG) and neutrophil activating peptide (NAP-2), which is the proteolytic derived end product. The temporal ocular expression of the chemokine PBP before and during corneal infection over several days by Pseudomonas aeruginosa was examined by immunohistochemistry. Prior to corneal infection, immunohistochemical staining demonstrated the constitutive expression of PBP in the cornea, lens and retina. PBP expression was present in the corneal epithelium, stromal fibroblasts and endothelium. There was a temporal increase in PBP expression in the cornea after infection. The entire cornea exhibited extensive cellular infiltration by positive PBP staining infiltrating cells within 6 days post-infection. The cornea, lens and retina underwent extensive degradation within 5-6 days post-infection with some apparent selective increase in PBP staining in the lens and retina.

Increased invasion through basement membrane by CXCL7-transfected breast cells.

BACKGROUND: CXC chemokines may modify breast cancer cells and surrounding extracellular matrix to facilitate metastasis. CXCL7 is heparin binding, has heparanase activity, and is a ligand to CXCR2, a G-protein-linked receptor. METHODS: Isogenic cell lines, malignant MCF10CA1a.cl1 cells, and premalignant MCF10AT cells were used. CXCR2 and CXCL7 expression levels were quantified by reverse transcriptionase-polymerase chain reaction and Western blot. MCF10AT cells were stably transfected with CXCL7, and matrigel invasion assays were performed. Antibody to CXCL7 was used to inhibit invasion. CXCL7 secretion by transfectants and heparanase activity were quantified by enzyme-linked immunosorbent assay. RESULTS: CXCL7 and CXCR2 expression were significantly higher in malignant MCF10CA1a.cl1 cells than in premalignant MCF10AT cells. Secreted CXCL7, secreted heparanase activity, and invasiveness were all increased in CXCL7-transfected MCF10AT cells. CXCL7 antibody inhibited invasion of CXCL7-transfected MCF10AT cells. CONCLUSIONS: Malignant MCF10CA1a.cl1 cells express more CXCL7 and CXCR2 than premalignant MCF10AT cells. CXCL7-transfected MCF10AT cells are as invasive as malignant breast cancer cells.

The Kolb learning cycle in American Board of Surgery In-Training Exam remediation: the Accelerated Clinical Education in Surgery course.

BACKGROUND: Surgical training integrates the 4 steps of the Kolb learning cycle. Residents who scored at 30th percentile or less on the American Board of Surgery In-Training Exam (ABSITE) were enrolled in the Accelerated Clinical Education in Surgery (ACES) course that incorporated the Kolb cycle. METHODS: For concrete experience, Surgical Education and Self-Assessment Program (SESAP-13) was completed according to the syllabus. For reflective consideration, further reading was done on SESAP 13 topics and corresponding ABSITE Keywords. For the abstract hypotheses step; these keywords and topics were reviewed with the mentor. Active testing involved a required weekly on-line quiz based on the syllabus. RESULTS: Correct scores on the ABSITE increased for 78.6% of residents in the ACES course, with 28.6% scoring 30th percentile or greater. Senior percent correct scores increased by 7.3% and junior percentile scores by 12.5%. CONCLUSIONS: Remediation using the Kolb cycle improved ABSITE performance for a majority of participants.

Expression patterns of lymphangiogenic and angiogenic factors in a model of breast ductal carcinoma in situ.

BACKGROUND: Foci of invasion are found in greater than 20% of excised specimens of breast ductal carcinoma in situ (DCIS). Since lymphangiogenesis markers are associated with the potential for increased lymph node metastasis, the purpose of the current study was to determine expression of lymphangiogenesis molecular markers in a model of aggressive DCIS. METHODS: From the MCF10A xenograft model, comedo type MCF10DCIS.com cells, premalignant MCF10AT, and invasive MCF10CA1a.cl1 cells were tested. Invasion was tested by Matrigel invasion assays (Becton-Dickinson, Bedford, MA). Gene expression was determined by reverse transcriptase-polymerase chain reaction and protein expression by immunoblot, normalized to beta-actin. RESULTS: MCF10DCIS.com cells were 4-fold more invasive than MCF10AT cells (P < .01), and expressed several-fold more mRNA and protein than MCF10AT and MCF10CA1a.cl1 cells for vascular endothelial growth factor C, vascular endothelial growth factor D, and lymphatic vessel endothelial hyaluronan receptor 1 (P < .01). CONCLUSIONS: A subset of comedo-type DCIS cells are invasive, and expression of lymphangiogenesis markers is greater at the mRNA and protein levels than by invasive cancer cells (P < .01). These additional molecular markers may characterize aggressive DCIS more precisely.

Murine ocular heparanase expression before and during infection with Pseudomonas aeruginosa.

PURPOSE: To demonstrate the constitutive expression and regulation of heparanase (heparan sulfate endoglycosidase) in the normal mouse eye and in mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naïve (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and corneal heparanase gene and protein expression were detected by semiquantitative RT-PCR and immunoblot analysis. Immunohistochemistry was also applied to characterize corneal heparanase in naïve mice. RESULTS: Heparanase mRNA and protein expression were detected in uninfected corneas of C57BL/6J mice. Immunohistochemical studies indicated heparanase protein expression was primarily in the corneal epithelium before corneal infection and was also in the corneal stroma after infection. Immunohistochemical studies of uninfected and infected whole eyes of naïve mice indicated heparanase protein expression in most layers of the retina, but the expression did not appear to be upregulated during corneal infection. Staining was most intense in the inner photoreceptor layer of the retina. CONCLUSIONS: Heparanase was constitutively expressed in both the corneal epithelium and several retinal layers before intracorneal infection with P. aeruginosa. Temporal upregulation of corneal heparanase protein expression was detected in naïve mice during infection, most likely due to heparanase positive infiltrating cells, but the protein was not upregulated in corneas from immunized mice because they had a lower inflammatory response, associated with the restoration of corneal clarity. There did not appear to be temporal upregulation of heparanase expression in the retina of infected mice, as determined by immunohistochemistry.

Can a practicing surgeon detect early lymphedema reliably?

BACKGROUND: Lymphedema may be identified by simpler circumference changes as compared with changes in limb volume. METHODS: Ninety breast cancer patients were prospectively enrolled in an academic trial, and seven upper extremity circumferences were measured quarterly for 3 years. A 10% volume increase or greater than 1 cm increase in arm circumference identified lymphedema with verification by a lymphedema specialist. Sensitivity and specificity of several different criteria for detecting lymphedema were compared using the academic trial as the standard. RESULTS: Thirty-nine cases of lymphedema were identified by the academic trial. Using a 10% increase in circumference at two sites as the criterion, half the lymphedema cases were detected (sensitivity 37%). When using a 10% increase in circumference at any site, 74.4% of cases were detected (sensitivity 49%). Detection by a 5% increase in circumference at any site was 91% sensitive. CONCLUSIONS: An increase of 5% in circumference measurements identified the most potential lymphedema cases compared with an academic trial.

Ethnicity related differences in the survival of young breast carcinoma patients.

BACKGROUND: African-American women face an increased risk of early-onset breast carcinoma compared to white American women, and breast carcinoma has been reported to be particularly aggressive in premenopausal women. METHODS: Surveillance, Epidemiology, and End Results Program data were analyzed for 507 African-American and 1378 white patients from Detroit diagnosed with breast carcinoma under the age of 40 between 1990 and 1999. RESULTS: The proportion of in situ disease detected in African-American patients between 1995 and 1999 nearly doubled compared to the 1990-1994 interval (11.3% compared to 6.4%) but was consistently lower than the proportion of in situ disease seen in white patients for the same intervals (15.7% and 16.4% respectively). Evaluation of patients with invasive disease revealed that African-American patients had larger mean tumor size (3.4 cm versus 2.6 cm; P < 0.001), lower rates of localized disease (42.4% versus 52.1%; P < 0.001), higher rates of estrogen receptor negativity (61.9% versus 44.4%; P < 0.001), and higher proportions of medullary tumors (5.8% versus 3.3%; P = 0.021). Cox proportional hazards survival analysis adjusted for age, tumor size, nodal status, hormone receptor status, and histology showed higher mortality rates for African-American patients at all disease stages. Relative risk of death for African-American patients was 1.94 in patients with localized disease (95% confidence interval [CI], 1.23-3.05), 1.58 for regional disease (95% CI = 1.18-2.11), and 2.32 for distant disease (95% CI = 1.15-4.69). CONCLUSIONS: These findings show that young African-American breast carcinoma patients face an increased mortality risk. Additional studies evaluating risk and treatment response in this subset of patients are warranted.

Multiple heparanases are expressed in polymorphonuclear cells.

BACKGROUND: Heparan sulfate proteoglycans are complex cell surface molecules containing polysaccharides called heparan sulfate. Lysosomes, platelet granules, and neutrophils (polymorphonuclear cells) contain heparanases that degrade heparan sulfate. There are at least two groups of heparanases: connective tissue-activating-peptide (CTAP-III) and mammalian heparanase (hpa). The purpose of this study was to quantify the expression of both CTAP-III and hpa in neutrophils and their heparanase activity. MATERIALS AND METHODS: Neutrophils were isolated from whole blood, total RNA collected, and reverse transcriptase--polymerase chain reaction (RT-PCR) performed. Primers were designed for CTAP-III and hpa-1 sequences from GenBank. Neutrophil lysate underwent Western blot analysis (and quantification) with antibodies to the C-terminus of CTAP-III and the 50-kDa subunit of hpa1. Chromatography separated these components of lysate, which were then tested for heparanase activity. RESULTS: Both CTAP-III (281 bp) and hpa-1 (485 bp) messenger RNA (mRNA) were expressed equally by neutrophils with use of quantitative RT-PCR. By Western blot analysis, a CTAP-III-like protein was detected at 80 kDa, and hpa-1 was detected as a 50-kDa protein, with expression not significantly different (P > 0.05). Heparanase activity was significantly different (P < 0.0001) for the 50-kDa hpa-1 protein (1.51 x 10(-6) micromol/min) and the 80-kDa CTAP-III-like protein (0.85 x 10(-6) micromol/min). CONCLUSIONS: Human neutrophils express mRNA and protein for both a CTAP-III-like protein and hpa-1. Although expressed in similar quantity for mRNA and protein, Hpa-1 was more active as heparanase than the CTAP-III-like protein. With more than one class of heparanase in their granules, neutrophils may be able to modify different kinds of heparan sulfate chains.