miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer.

BACKGROUND: microRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown. RESULTS: In the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes. CONCLUSION: Loss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer.

Embedded Mental Health: Promotion of Psychological Hygiene Within a Submarine Squadron.

INTRODUCTION: Psychological fitness is an important component to operational unit readiness and success. Embedding behavioral health providers can reduce unplanned personnel losses (UPL) as a result of psychological stress. The U.S. Submarine Forces implemented the Submarine Squadron 6 (CSS-6) Embedded Mental Health Pilot (EMHP) Program to address this type of UPL, which is classified as a Code 2 loss. The aim of the study is to evaluate the effectiveness of the EMHP Program at reducing UPL by improving psychological readiness through expedited access to care. MATERIALS AND METHODS: Using data from the CSS-6 EMHP Program, we identified the cohort of patients who were evaluated and received a full course of treatment from August 1, 2013, to April 30, 2015, and examined their final dispositions. A comparative review of Code 2 losses between 2012 and 2014 was performed to assess for any reduction in the annual incidence of Code 2 losses with EMHP. The Outcome Questionnaire (OQ-45) survey was used to determine the quantitative impact of EMHP on patient psychological readiness. We performed multiple regression analysis to identify any significant correlation between all independent variables and improvement in final OQ-45 scores. We performed logistic regression analysis to assess the logistic score as a function of predicting patient probability of returning vice not returning to duty. The logistic score is a by-product of the end results data and was not an original metric when the program was started. The Clinical Investigations Department at Naval Medical Center, Portsmouth waived this study from institutional review board review. Authorization was obtained from the U.S. Submarine Forces Command Public Affairs Office to publish the contents of this study. RESULTS: EMHP providers conducted a total of 878 patient sessions for 183 sailors over a 21-month period. There were eight fewer Code 2 losses after 2014, the first full calendar year with EMHP. This decrease in the number of Code 2 losses was in fact statistically significant, given p < 0.001. EMHP providers saw a 200% increase in patient volume and contributed to a 12% decrease in the annual incidence of Code 2 losses in 2014. Seventy patients returning to duty demonstrated clinically and statistically significant improvements in OQ-45 scores at the end of treatment. Only the initial symptomatic distress score on the OQ-45 survey demonstrated any statistical significance of predicting an improvement in OQ-45 composite scores by the end of treatment, given p < 0.01. A negative logistic score was significantly associated with not returning to duty (odds ratio, 16.0; 95% confidence interval, 5.22-49.02; χ2 = 30.63; p < 0.001). CONCLUSION: The EMHP Program reduced Code 2 losses and positively promoted psychological hygiene for submariners. With the establishment of embedded programs at other squadrons, we can develop a longitudinal study that provides a more inclusive assessment of this model. A future study may be warranted to evaluate the validity of the logistic score as a metric to determine further fitness for submarine duty.

Increased expression levels of WAVE3 are associated with the progression and metastasis of triple negative breast cancer.

BACKGROUND: Breast Cancer (BC) is a heterogeneous disease comprised of at least five genetically distinct subtypes, which together form the second leading cause of cancer death in women in the United States. Within BC subtypes, those classified as Triple Negative BCs (TNBCs) exhibit dismal survival rates due to their propensity to develop distant metastases. We have identified the WAVE3 protein, which is a critical regulator of actin cytoskeleton dynamics that are required for the motility and invasion of cancer cells through its activation of the Arp2/3 complex, as a key regulator of the different steps of the invasion-metastasis cascade in BC, especially in the more aggressive TNBCs. Our published studies have also shown that elevated expression levels of WAVE3 in the TNBC cell lines directly contribute to their increased invasion and metastasis potentials both in vitro and in vivo in murine models of BC metastasis. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we utilized both immunohistochemistry (IHC) of primary human BC tumors as well as quantitative real-time RT-PCR of WAVE3 in the peripheral blood of BC patients to clearly establish that WAVE3 is a predictive marker of overall BC patients' survival. High levels of WAVE3 were predictive for reduced distant recurrence-free survival as well as for decreased disease-specific mortality. Our analysis of WAVE3 expression levels in the peripheral blood of BC patients showed that WAVE3 is highly expressed in the blood of patients who developed metastatic breast cancer compared to those who did not. WAVE3 expression was also highly upregulated in the blood of BC patients with the more aggressive TNBC subtype. CONCLUSIONS: Together, these findings establish WAVE3 as a novel marker for increased risk of breast-cancer-specific mortality and for the metastatic potential of the TNBCs, and also identify WAVE3 as an attractive therapeutic target for the treatment of metastatic BC.

miR-31 is a broad regulator of ß1-integrin expression and function in cancer cells.

Integrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathologic conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs (miRNA) has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell-based, genetic, biochemical, flow cytometry, and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5, and αV) of ß1 integrins and also ß3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV, and ß3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3' untranslated region of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. Although different reports have shown that a single integrin can be regulated by several miRNAs, here we show that a single miRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis.

Optimizing a District of Columbia Strategic National Stockpile dispensing center.

On October 4, 2003, the District of Columbia Department of Health (DOH) held a Strategic National Stockpile (SNS) exercise designed to test its plan for operating mass dispensing centers during a bioterrorist attack or other emergency. The main goals of the exercise were to maximize the throughput of the dispensing plan and improve dispensing procedures. These goals are important for quantifying the resources (eg, numbers and types of staff) necessary to respond to different types and sizes of events, as well as for minimizing the potential for errors or confusion in dispensing medications. We set up the dispensing center according to the District's SNS plan and recruited volunteers to role-play potentially exposed residents. During the exercise, we collected detailed data on the service times for each step in the dispensing process. We also collected observations from exercise participants and observers. We found that the DOH dispensing center could achieve a throughput of 2.5 persons per minute. Using computer modeling, we recommended changes to the dispensing plan that would enable it to achieve a higher throughput of four to five persons per minute. Other recommendations addressed improvements to dispensing plans and procedures.

Calmodulin binds to the cytoplasmic domain of angiotensin-converting enzyme and regulates its phosphorylation and cleavage secretion.

The rate of cleavage secretion of the enzymatically active ectodomain of angiotensin-converting enzyme (ACE) is regulated by tyrosine phosphorylation of the protein and by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Here, we report that both calmodulin inhibitor (CaMI) and calmodulin kinase inhibitor could also enhance cleavage secretion of ACE. This effect was accompanied by the dissociation of calmodulin from a specific region within the cytoplasmic domain of ACE to which it had been bound. The same domain of ACE was phosphorylated, and both CaMI and PMA caused dephosphorylation of ACE as well. Mass spectrometric and mutational analyses identified Ser730 as the only phosphorylated residue in the cytoplasmic domain of ACE. The Ser730 --> Ala mutant of ACE was not phosphorylated, but it still bound calmodulin, and its cleavage secretion was enhanced by both CaMI and PMA. Similarly, when Ser730 was replaced by the phosphoserine mimetic, Asp, cleavage secretion of the resultant mutant remained susceptible to the enhancing effect of CaMI and PMA. These results demonstrate that, although CaMI and PMA can enhance both cleavage secretion of ACE and its dephosphorylation, the two effects are not mutually interdependent.

Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme.

Both germinal (gACE) and somatic (sACE) isozymes of angiotensin-converting enzyme (ACE) are type I ectoproteins whose enzymatically active ectodomains are cleaved and shed by a membrane-bound protease. Here, we report a role of protein tyrosine phosphorylation in regulating this process. Strong enhancements of ACE cleavage secretion was observed upon enhancing protein Tyr phosphorylation by treating gACE- or sACE-expressing cells with pervanadate, an inhibitor of protein Tyr phosphatases. Secreted gACE, cell-bound mature gACE and its precursors were all Tyr-phosphorylated, as was the endoplasmic reticulum protein, immunoglobulin heavy chain-binding protein, that co-immunoprecipitated with ACE. The enhancement of cleavage secretion by pervanadate did not require the presence of the cytoplasmic domain of ACE, and it was not accomplished by enhancing the rate of intracellular processing of the protein. The observed enhancement of cleavage secretion of ACE in pervanadate-treated cells was specifically blocked by an inhibitor of the p38 mitogen-activated protein (MAP) kinase but not by inhibitors of many other Ser/Thr and Tyr protein kinases, including a specific inhibitor of protein kinase C that, however, could block the enhancement of cleavage secretion elicited by phorbol ester. These results indicate that ACE Tyr phosphorylation, probably in the endoplasmic reticulum, enhances the rate of its cleavage secretion at the plasma membrane using a regulatory pathway that may involve p38 MAP kinase.