Search

Background: Triple-negative breast cancer (TNBC) comprises 10%-20% of invasive breast carcinoma cases. Patients with TNBC exhibit poorer 5-year survival and higher rates of metastasis and recurrence than patients with other breast cancer subtypes. TNBC is further divided based on gene expression profiling into androgen receptor (AR)-positive (AR+) TNBC and AR-negative TNBC (or quadruple-negative breast cancer [QNBC]). While AR is an emerging therapeutic target for AR+ TNBC, there are no treatment options for QNBC. AR expression is associated with favorable disease-free, overall, and recurrence-free survival in patients with TNBC, suggesting that AR loss in TNBC tumors confers an aggressive disease course. Genomic instability has been recognized as one of the drivers of tumorigenesis and can be evidenced by the presence of chromosomal instability, such as DNA copy number alterations (CNAs). Although there are many studies characterizing the genomic profiles of TNBCs, little is known about the tumor biology and genetic makeup of QNBCs. In this study, we explored the impact of CNAs on microRNA (miRNA) expression and, subsequently, on signaling pathways associated with QNBC aggressiveness and clinical outcomes. Methods: AR expression was immunohistochemically assessed in tissues of 53 patients with TNBC from the Histopathology and Tissue Shared Resources of Georgetown University. AR positivity was defined as AR expression in >1% of cells. Genome-wide copy number profiling of formalin-fixed paraffin-embedded samples was performed using the Agilent SurePrint G3 Human CGH Microarray, and miRNA expression analysis was conducted using NanoString nCounter Human v3a miRNA Expression Assay. Differentially expressed miRNAs between TNBC and. QNBC samples were integrated with array CGH data from the same tissue sample to identify common gene targets that may be affected by both CNAs and miRNA deregulation. Functional enrichment analysis was performed to identify cancer pathways deregulated in QNBC. The expression levels of Centrosome Amplification 20 (CA20) and Chromosome Instability 25 (CIN 25) gene signatures were evaluated in 524 primary invasive breast cancers from The Cancer Genome Atlas (TCGA) database. Results: We observed that 64% of TNBC samples lacked AR expression and that QNBCs exhibited a significantly higher CNA level (p50% of QNBCs. Additionally, CNAs affecting genes of the CIN25 signature were significantly higher in QNBC than in TNBC; however, CNAs in CA20 signature genes were similar in QNBC and TNBC samples. Notably, the expression levels of CA20 and CIN25 genes were significantly higher in QNBC than in TNBC. Moreover, we identified 184 differentially expressed miRNAs between QNBC and TNBC samples (p Citation Format: Shristi Bhattarai, Bruna M. Sugita, Luciane Cavalli, Ritu Aneja. Androgen receptor loss is associated with genomic instability characterized by copy number alterations and mirna deregulation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-13.

MiR-150-5p is frequently deregulated in cancer, with expression and mode of action varying according to the tumor type. Here, we investigated the expression levels and role of miR-150-5p in the aggressive breast cancer subtype triple-negative breast cancer (TNBC). MiR-150-5p expression levels were analyzed in tissue samples from 113 patients with invasive breast cancer (56 TNBC and 57 non-TNBC) and 41 adjacent non-tumor tissues (ANT). Overexpression of miR-150-5p was observed in tumor tissues compared with ANT tissues and in TNBC compared with non-TNBC tissues. MiR-150-5p expression levels were significantly associated with high tumor grades and the Caucasian ethnicity. Interestingly, high miR-150-5p levels were associated with prolonged overall survival. Manipulation of miR-150-5p expression in TNBC cells modulated cell proliferation, clonogenicity, migration, and drug resistance. Manipulation of miR-150-5p expression also resulted in altered expression of its mRNA targets, including epithelial-to-mesenchymal transition markers, MYB, and members of the SRC pathway. These findings suggest that miR-150-5p is overexpressed in TNBC and contributes to the aggressiveness of TNBC cells in vitro.

Triple negative breast cancer (TNBC), a clinically aggressive breast cancer subtype, affects 15–35% of women from Latin America. Using an approach of direct integration of copy number and global miRNA profiling data, performed simultaneously in the same tumor specimens, we identified a panel of 17 miRNAs specifically associated with TNBC of ancestrally characterized patients from Latin America, Brazil. This panel was differentially expressed between the TNBC and non-TNBC subtypes studied (p ≤ 0.05, FDR ≤ 0.25), with their expression levels concordant with the patterns of copy number alterations (CNAs), present mostly frequent at 8q21.3-q24.3, 3q24-29, 6p25.3-p12.2, 1q21.1-q44, 5q11.1-q22.1, 11p13-p11.2, 13q12.11-q14.3, 17q24.2-q25.3 and Xp22.33-p11.21. The combined 17 miRNAs presented a high power (AUC = 0.953 (0.78–0.99);95% CI) in discriminating between the TNBC and non-TNBC subtypes of the patients studied. In addition, the expression of 14 and 15 of the 17miRNAs was significantly associated with tumor subtype when adjusted for tumor stage and grade, respectively. In conclusion, the panel of miRNAs identified demonstrated the impact of CNAs in miRNA expression levels and identified miRNA target genes potentially affected by both CNAs and miRNA deregulation. These targets, involved in critical signaling pathways and biological functions associated specifically with the TNBC transcriptome of Latina patients, can provide biological insights into the observed differences in the TNBC clinical outcome among racial/ethnic groups, taking into consideration their genetic ancestry.

Background To investigate the global expression profile of miRNAs, their impact on cellular signaling pathways, and their association with poor prognostic parameters in African-American (AA) patients with triple negative breast cancer (TNBC). Methods Twenty-five samples of AA TNBC patients were profiled for global miRNA expression and stratified considering three clinical-pathological parameters: tumor size, lymph node (LN), and recurrence (REC) status. Differential miRNA expression analysis was performed for each parameter, and their discriminatory power was determined by Receiver Operating Characteristic (ROC) curve analysis. KMplotter was assessed to determine the association of the miRNAs with survival, and functional enrichment analysis to determine the main affected pathways and miRNA/mRNA target interactions. Results A panel of eight, 23 and 27 miRNAs were associated with tumor size, LN, and REC status, respectively. Combined ROC analysis of two (miR-2117, and miR-378c), seven (let-7f-5p, miR-1255b-5p, miR-1268b, miR-200c-3p, miR-520d, miR-527, and miR-518a-5p), and three (miR-1200, miR-1249-3p, and miR-1271-3p) miRNAs showed a robust discriminatory power based on tumor size (AUC = 0.917), LN (AUC = 0.945) and REC (AUC = 0.981) status, respectively. Enrichment pathway analysis revealed their involvement in proteoglycans and glycan and cancer-associated pathways. Eight miRNAs with deregulated expressions in patients with large tumor size, positive LN metastasis, and recurrence were significantly associated with lower survival rates. Finally, the construction of miRNA/mRNA networks based in experimentally validated mRNA targets, revealed nodes of critical cancer genes, such as AKT1, BCL2, CDKN1A, EZR and PTEN. Conclusions Altogether, our data indicate that miRNA deregulated expression is a relevant biological factor that can be associated with the poor prognosis in TNBC of AA patients, by conferring to their TNBC cells aggressive phenotypes that are reflected in the clinical characteristics evaluated in this study.

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).

Triple-negative breast cancer (TNBC) can be further classified into androgen receptor (AR)-positive TNBC and AR-negative TNBC or quadruple-negative breast cancer (QNBC). Here, we investigated genomic instability in 53 clinical cases by array-CGH and miRNA expression profiling. Immunohistochemical analysis revealed that 64% of TNBC samples lacked AR expression. This group of tumors exhibited a higher level of copy number alterations (CNAs) and a higher frequency of cases affected by CNAs than TNBCs. CNAs in genes of the chromosome instability 25 (CIN25) and centrosome amplification (CA) signatures were more frequent in the QNBCs and were similar between the groups, respectively. However, expression levels of CIN25 and CA20 genes were higher in QNBCs. miRNA profiling revealed 184 differentially expressed miRNAs between the groups. Fifteen of these miRNAs were mapped at cytobands with CNAs, of which eight (miR-1204, miR-1265, miR-1267, miR-23c, miR-548ai, miR-567, miR-613, and miR-943), and presented concordance of expression and copy number levels. Pathway enrichment analysis of these miRNAs/mRNAs pairings showed association with genomic instability, cell cycle, and DNA damage response. Furthermore, the combined expression of these eight miRNAs robustly discriminated TNBCs from QNBCs (AUC = 0.946). Altogether, our results suggest a significant loss of AR in TNBC and a profound impact in genomic instability characterized by CNAs and deregulation of miRNA expression.

Cryptococcosis is a systemic mycosis with a chronic or subacute progression caused by the inhalation of dehydrated yeasts or basidiospores. The causative agents are C. gattii and C. neoformans. The latter is more commonly associated with cellular immunodeficiency and is not rare in patients with Acquired Immunodeficiency Syndrome (AIDS). Cryptococcosis is common in pregnant women with AIDS; however, it is uncommon for the placenta to be affected, with few reported cases in the literature. We present the case of a pregnant woman with AIDS who had placental and pulmonary cryptococcosis associated with fungemia, with a satisfactory clinical outcome obtained after therapy.

// Bruna Sugita 1 , Mandeep Gill 2 , Akanskha Mahajan 2 , Anju Duttargi 2 , Saurabh Kirolikar 2 , Rodrigo Almeida 1 , Kenny Regis 2 , Olusayo L. Oluwasanmi 2 , Fabio Marchi 3 , Catalin Marian 4, 8 , Kepher Makambi 2, 5 , Bhaskar Kallakury 6 , Laura Sheahan 7 , Iglenir J.Cavalli 1 , Enilze M. Ribeiro 1 , Subha Madhavan 2, 7 , Simina Boca 2, 7 , Yuriy Gusev 2, 7 , Luciane R. Cavalli 2 1 Department of Genetics, Federal University of Parana, Curitiba, PR, Brazil 2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 3 International Research Center-CIPE, A. C. Camargo Cancer Center, Sao Paulo, SP, Brazil 4 The Ohio State University Comprehensive Cancer Center, Division of Cancer Prevention and Control, College of Medicine, The Ohio State University, Columbus, Ohio 5 Departments of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University, Washington, DC USA 6 Department of Pathology, Georgetown University Medical Center, Washington, DC, USA 7 Innovation Center for Biomedical Informatics, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 8 The University of Medicine and Pharmacy Timisoara, Timisoara, Romania Correspondence to: Luciane R. Cavalli, email: lrc@georgetown.edu Keywords: microRNA, triple negative breast cancer, African American, non-Hispanic white, copy number Received: July 02, 2016 Accepted: October 25, 2016 Published: November 02, 2016 ABSTRACT Triple Negative Breast Cancer (TNBC), a clinically aggressive subtype of breast cancer, disproportionately affects African American (AA) women when compared to non-Hispanic Whites (NHW). MiRNAs(miRNAs) play a critical role in these tumors, through the regulation of cancer driver genes. In this study, our goal was to characterize and compare the patterns of miRNA expression in TNBC of AA ( n = 27) and NHW women ( n = 30). A total of 256 miRNAs were differentially expressed between these groups, and distinct from the ones observed in their respective non-TNBC subtypes. Fifty-five of these miRNAs were mapped in cytobands carrying copy number alterations (CNAs); 26 of them presented expression levels concordant with the observed CNAs. Receiving operating characteristic (ROC) analysis showed a good power (AUC ≥ 0.80; 95% CI) for over 65% of the individual miRNAs and a high combined power with superior sensitivity and specificity (AUC = 0.88 (0.78−0.99); 95% CI) of the 26 miRNA panel in discriminating TNBC between these populations. Subsequent miRNA target analysis revealed their involvement in the interconnected PI3K/AKT, MAPK and insulin signaling pathways. Additionally, three miRNAs of this panel were associated with early age at diagnosis. Altogether, these findings indicated that there are different patterns of miRNA expression between TNBC of AA and NHW women and that their mapping in genomic regions with high levels of CNAs is not merely physical, but biologically relevant to the TNBC phenotype. Once validated in distinct cohorts of AA women, this panel can potentially represent their intrinsic TNBC genome signature.

Breast cancer is the most common and the leading cause of female mortality among South African (SA) women. Several non‑biological and biological risk factors may be attributed to their observed high mortality rate; however, the molecular profiles associated with their breast tumors are poorly characterized. The present study examined the patterns of genome-wide copy number alterations (CNAs) and their potential impact on functional cellular pathways targeted by cancer driver genes in patients with breast cancer from the Western Cape region of SA. Array-comparative genomic hybridization analysis, performed in 28 cases of invasive breast cancer, revealed a mean number of 8.68±6.18 CNAs per case, affecting primarily the Xp22.3 and 6p21-p25 cytobands (57.14% of the cases), followed by 19p13.3-p13.11 (35.7%), 2p25.3-p24.3, 4p16.3-p15.3, 8q11.1-q24.3 and 16 p13.3-p11.2 (32.14%). Functional enrichment analysis of genes and microRNA targets mapped in these affected cytobands revealed critical cancer-associated pathways, including fatty acid biosynthesis and metabolism, extracellular matrix-receptor interaction, hippo and tumor protein p53 signaling pathways, which are regulated by known cancer genes, including CCND1, CDKN1A, MAPK1, MDM2, TP53 and SMAD2. An inverse correlation was observed among the number of CNAs and tumor size and grade; CNAs on the 4p and 6p cytobands were also inversely correlated with tumor grade. No association was observed in the number of CNAs and/or the affected cytobands and the different ethnic groups of the SA patients, indicating that their tumor genome is affected by CNAs, irrespectively of their genetic descent. Additional genomic tumor profiling in SA and other Sub-Saharan African patients with breast cancer is required to determine the associations of the CNAs observed with prognosis and clinical outcome.

MiRNA dysregulation has been linked to cancer initiation and progression, through their control in multiple cancer associated signaling pathways. MiRNA low specificity of target interaction confer them the ability to cooperatively regulate a single biological process. Epithelial mesenchymal transition (EMT) plays pivotal roles in cancer progression and metastasis, through the regulation of transcription factors and proteins, such as E-cadherin, Slug, Snail, Twist and Zeb1. This process is also controlled post-transcriptionally by a complex and regulated network of miRNAs. We have shown that miR-150-5p and miR-661, up-regulated in triple negative breast cancer (TNBC) when compared to normal tissue and other breast cancer subtypes, exert an oncogenic action and modulate cell proliferation, clonogenicity, migration and cytotoxic response in in vitro TNBC models. The main objective of this study was to determine whether the modulation of these phenotypes were due to the regulation of their corresponding gene targets associated with EMT. By conducting an integrated computational and experimental analysis, in which miR-150-5p and miR-661 were suppressed or ectopically expressed in transfected MDA-MB-231 cells, we showed that they present common regulatory roles in cell adhesion and motility signaling networks, including those involved in EMT. Gene expression analysis in a targeted panel of 770 genes with primary functional annotations in EMT, cell adhesion and motility and metastasis, revealed that: i. the suppression of miR-150-5p and miR-661, affected the expression of 160 and 123 genes, respectively; 18% of these genes were commonly affected by both miRNAs, including ACVR1C (ALK-7), CHAD, GIMAP6, SFRCL1, SPARCL1 and TBX1; ii. the ectopic expression of miR-150-5p and miR-661, affected the expression of 283 and 267 genes, respectively; 27% of which commonly affected by both miRNAs, including AKT1, DCL1, MAPK2, MMP1, SFRP1 and TPM2. iii. 26 genes were inversely affected by the suppression and ectopic expression of miR-150-5p and miR-661 (consistent with the general post-transcriptional regulation of miRNA/mRNA targets); 73% of these genes were predicted to be miRs150-5p and 661 direct or indirect targets. In summary, based on the computational and experimental data presented, we provide evidences that strongly suggest that miRNA-150-5p and miR-661 modulate the aggressive tumor phenotype in TNBC cells by regulating common gene targets associated with the EMT process. Further functional studies in TNBC experimental models will allow us to dissect the mechanisms by which they contribute to the TNBC aggressive phenotypes by synergistically regulating EMT. Funding: Partially funded by Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), CNPq, Capes-Brazil (scholarship to PMMO, BMS, ASF). Citation Format: Patricia MM Ozawa, Bruna M. Sugita, Aline S. Fonseca, Yara Rodriguez, Yuriy Gusev, Paolo Fadda, Deepak Kumar, Luciane R. Cavalli. The synergistic role of miR-150-5p and miR-661 in regulating epithelial mesenchymal transition in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 496.

Epidemiologic and molecular studies have shown that breast cancer subtypes are distributed unevenly among racial/ethnic groups. The incidence of the triple-negative breast cancer (TNBC) subtype in particular, one of the most clinically aggressive breast cancer subtypes, affects around 20% of women from Latin America, 24-28% of African-Americans, 6-8% of Asians, and around 12% of non-Hispanic Whites. In general Latin American patients with TNBC are more often diagnosed with earlier age, advanced stage, are likely to experience metastasis and be refractory to treatment. Although the characterization of the genomic profiles in each breast cancer subtype has been extensively performed, few studies have characterized them in specific ethnic groups. This translates to a deficiency in the understanding of the intrinsic characteristics of their tumors' genome, which can differentially impact their tumor phenotypes and clinical behavior. In this study we performed genome-wide array-CGH and miRNA profiling in TNBC (n=29) and non-TNBC cases (n=27) of patients from Latin America, Brazil (Curitiba-PR), to characterize their patterns of copy number and miRNA expression. These data were integrated to determine whether the location of miRNAs in genomic regions rich in copy number alterations (CNAs) could impact their expression levels and whether there were common miRNA target genes affected by both CNAs and miRNA deregulation. Our findings showed a distinct pattern of CNAs and miRNA expression between the TNBC and non-TNBC cases. A total number of 309 and 124 CNAs were identified in the TNBC and non-TNBC cases, with an average of 10.65 and 7.75 CNAs per case, respectively. In the TNBC cases, the most frequent (>30% of the cases) cytobands affected by CNAs were 8q11.21-q24.3, 1q21.1-q44, 7q11.23-q36.3, 12p13.33-p11.1, 3q11.2-q29, and 6p25.3-p11.2. MiRNA profiling analysis showed 209 miRNAs with significant differentially expression between these two groups (P0.7 was observed for 75% of these miRNAs; miRs-567, 610, 802, 944 and 1260a presented the highest discriminatory power, with AUC values >0.8. A number of 1312 genes were identified mapped in the most common CNAs and targeted by these miRNAs. Subsequent pathway analysis of the 12 miRNAs showed their involvement in cancer-related pathways, including the Hippo, TGF-beta, and Ras signaling pathways; a close hierarchical cluster was observed with the miRs-592, 634, 802, 944, and 1260 in regulating these pathways. In conclusion, the TNBC and non-TNBC cases of the patients of this study presented a distinct pattern of CNAs and miRNA expression levels. The copy number and miRNA integrated analysis performed delineated the boundaries of genomic instability regions and the mapping of the most relevant miRNA targets in these TNBC cases, that can act as potential oncogenes and/or tumor suppressor genes in these regions. These findings contributed to the identification of unique ethnic miRNA/mRNA pairings and their corresponding cancer signaling pathways that are of relevance to the TNBC of Latin American patients and that can be directly and more efficiently targeted for therapy. Support: Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration. This research does not necessarily reflect the views of the FDA. The authors thank CNPq and CAPES, Brazil, for scholarships (B.S., S.R.P.). Note: This abstract was not presented at the conference. Citation Format: Bruna Sugita, Silma R. Pereira, Rodrigo Almeida, Rubens S. Lima, Cicero A. Urban, Simina Boca, Yuriy Gusev, Iglenir J. Cavalli, E.M.S.F. Ribeiro, Luciane R. Cavalli. Copy number and miRNA profiling in triple-negative breast cancer patients from Latin America [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A28.

In many cases, single-break vacuum circuit breakers are used as circuit breakers, and so the characteristics of double-break vacuum circuit breakers have not received sufficient attention. This report describes our investigations of the interruption characteristics of double-break vacuum circuit breakers. We have investigated and clarified the relationship between the voltage distribution ratio and the post arc current in double-break vacuum circuit breakers by measuring the post arc current in the single-break vacuum circuit breaker, the voltage distribution ratio, and the post arc current in double-break vacuum circuit breakers. Our investigation has shown that the post arc current scatter of the spiral contact is much larger than that of the axial magnetic field contact. This result shows that the bias of the voltage distribution ratio was caused by the imbalanced post arc current in each vacuum interrupter in the double-break vacuum circuit breaker. Therefore, we conclude that axial magnetic field contacts are suitable for double-break vacuum circuit breakers.

It is important to understand the interruption phenomena for contact materials in order to adapt them for use in high voltage. Accordingly, we have carried out measurements of the arc energy and anode surface temperature and observed the vacuum arc in a vacuum chamber. We have used three kinds of contact materials in this investigation, namely, Cu, CuCr, and AgWC. In addition, it is effective to develop a new contact material as the means of application to high voltage of vacuum circuit breaker. Therefore, we made a prototype of new contacts, based on the tests results, for adaptation to high-voltage vacuum circuit breakers. In addition, we carried out interruption tests using these contact materials.

Triple negative breast cancer (TNBC) disproportionately afflicts young African American (AA) women when compared to Non-Hispanic Whites (NHW), being usually diagnosed in advanced stage, likely to experience metastasis and often unresponsive to treatment. An increased number of studies have characterized the differences in the tumor biology between AA and NHW patients and have shown that breast tumors from AA patients present increased cell proliferation, elevated expression of angiogenesis markers and higher migration and invasive properties, the fundamental hallmarks of cancer. MiRNAs are short sequences of non-coding RNAs that act on gene expression regulation. In this study, our main objective was to determine the expression levels of miRNA-661 in the TNBC of AA when compared to the NHWs and to verify it is directly role in affecting the aggressive TNBC phenotype. MiRNA-661 expression analysis was conducted by RT-qPCR in the tumor tissue of 31 AA-TNBC patients and 17 NHW-TNBC patients. AA and NHW samples of 40 and 20 non-TNBC subtype, respectively, and 49 adjacent normal tissue (ANTs) were used as control for the subtype and tumor specificity, respectively. Our results showed a significant down-regulation of miR-661 expression in TNBC of AA in relation to the NWH (P Citation Format: Aline S. Fonseca, Selene Elifio-Esposito, Marilesia F. Souza, Akanksha Mahajan, Yara R. Zabala, Bruna M. Sugita, Luciane R. Cavalli. Regulatory role of miRNA-661 in triple-negative breast cancer of African American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1465. doi:10.1158/1538-7445.AM2017-1465

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that confers disease recurrence, treatment resistance and high mortality rates. MicroRNAs are a class of noncoding RNAs, that when dysregulated, impact tumorigenesis through the control of the expression of multiple mRNA targets involved in critical cancer signaling pathways. MiR-150-5p has been shown to control the expression of several driver oncogenes and/or tumor suppressor genes involved in these critical pathways. Its pattern of expression varies according to the cancer type; it has been observed mostly downregulated in hematological diseases and GI cancers, and upregulated in hormonal dependent cancers, such as prostate and breast cancer. The main objective of this study was to assess the patterns of expression of miR-150-5p in TNBC and determine its functional role in affecting the tumor phenotype. Archived paraffin samples of 113 patients with ductal breast carcinoma (56 of the TNBC and 57 of the non-TNBC subtype) and 49 adjacent normal tissue (ANT), obtained from the the pathology tumor bank of Lombardi Comprehensive Cancer Center, Washington DC, were profiled for miRNA using the wide-genome Nanostring platform and a Taqman specific miRNA-150-5p assay. Significant overexpression levels of miRNA-150-5p were observed in the tumor tissues when compared to the ANT and in the TNBC cases when compared to the non-TNBC cases, demonstrating its tumor and TNBC subtype specificity, respectively. Overexpressed levels of miRNA-150-5p were also preferentially observed in the TNBC cases from patients that presented with LN metastasis and breast cancer recurrence, indicating its association with poor prognosis. Interestingly, the TNBC of African-American patients, which is the ethnic group mostly affected by this cancer subtype, presented overexpression levels of this miRNA when compared to the Non-Hispanic White patients. Functional analysis performed in the TNBC cell lines, MDA-MB-231 and HCC1806, showed after transfection with miR-150-5p inhibitor, reduced levels on cell proliferation, clonogenicity, migration, drug resistance and expression of the EMT promoter markers, SLUG and SNAIL. These findings, indicate an oncogenic type of action of miRNA-150-5p in TNBC. In summary, miRNA-150-5p is upregulated in TNBC clinical cases in association with poor prognostic parameters and its functional inhibition, directly confers to the cells a reduction of their tumorigenic phenotype. Funding: This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA. Scholarship to B.S. was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Citation Format: Bruna M. Sugita, Yara Zabala, Aline Fonseca, Rodrigo Almeida, Yuriy Gusev, Simina Boca, Iglenir J. Cavalli, Enilze M. Ribeiro, Luciane R. Cavalli. The oncogenic role of miR-150-5p in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2017-3431

Triple negative breast cancer (TNBC) is a clinically and molecularly heterogeneous disease, which incidence and outcome vary among the different ethnic and racial groups. These tumors are more commonly seen in younger women of African and Latinas/Hispanic descents, usually diagnosed at more advanced stages, with non-localized disease. Molecular studies have shown differences in the biology of these tumors in Latinas as key contributors for high mortality. The main objective of our study was to identify a miRNA signature associated with TNBC of Latina patients by integrating miRNA and array-CGH data. Archived paraffin samples of 32 TNBC and 25 non-TNBC cases from Latina patients, obtained from the Clinical Hospital (UFPR) in Brazil, were profiled for miRNA and array-CGH using the Nanostring and the Agilent SurePrint G3 Human array-CGH platforms, respectively. The miRNA and array-CGH data, obtained in the same samples, was directly integrated and combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify the most relevant miRNAs and their corresponding gene targets. Eight-nine miRNAs were observed differentially expressed between the TNBC and non-TNBC lesions. Using miRBase and MiRDB target prediction databases 3,378 miRNAs targets were identified. After integration with array-CGH, a number of 15 miRNAs presented concomitant DNA copy number and miRNA alterations, reducing the number of targets to 1,242. Ingenuity pathway analysis identified the most affected canonical pathways, including IL-8, TGF-beta, PI3K-AKT and HER2 signaling pathways. Using our integration approach we were able to identify a robust miRNA signature associated with TNBC of Latina patients and to identify the potential molecular mechanism (s) that underline the observed miRNA deregulation in these patients. This signature revealed miRNAs and corresponding targets biologically relevant, involved in critical cancer signaling pathways and gene networks. Once this signature is functionally validated, its direct role in the aggressive clinical phenotype of these tumors will be determined. In summary, the findings of our study contributes to the identification of race/ethnic specific molecular targets that can set the basis for new TNBC treatments and for the future design of the most appropriate clinical trials and cancer control interventions for Latina women. Funding:This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI; U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA. Citation Format: Bruna M. Sugita, Mandeep Gill, Silma R. Pereira, Yara R. Zabala, Aline S. Fonseca, Selene E. Esposito, Catalin Marian, Iglenir J. Cavalli, Enilze MF Ribeiro, Yuriy Gusev, Luciane R. Cavalli. Integrated analysis of copy number and miRNA profiling in triple negative breast cancer of Latina women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1944.

The purpose of this study was to determine the feasible adjuvant therapy administration schedule of S-1 for locoregionally advanced squamous cell carcinoma of the head and neck (SCCHN). Patients receiving definitive treatments were randomly assigned to either arm A (51 cases) receiving oral S-1 of 2-week administration followed by 1-week rest for 6 months, or arm B receiving S-1 of 4-week administration followed by 2-week rest for 6 months. Planned treatment was given in 40% of patients in arm A and 29% in arm B. The cumulative rates of the relative total administration dose of S-1 at 100% were 54.9% (95% CI: 40.1–69.7%) in arm A and 34.3% (95% CI: 21.1–47.4%) in arm B, respectively (P=0.054). Adverse events were recorded in 41 patients (82.0%) in arm A and 48 patients (94.1%) in arm B (P=0.060). The incidences of diarrhoea (10 vs 28%; P

Osmotic shock transiently induces a volume change in the cells, followed by a restoration of the cell volume due to intracellular water regulation. Effect of long-term osmotic shock on the water regulation is not completely understood. Vacuole formation by long-term osmotic shock was investigated to clarify the water exclusion mechanism from cytoplasm into intracellular vacuoles in 3T3-L1 cells. Incubation of cells in hypotonic solution reversibly induced the vacuole formation. Staining of vacuoles with fluorescent dyes revealed that vacuoles were derived from endoplasmic reticulum and Golgi apparatus but not lysosomes. Membrane-impermeable fluorescent dyes were taken up into some vacuoles from cytoplasm and extracellular solution, suggesting that some vacuoles exhibit the dynamic changes for the connection of plasma membrane, and that transporter for membrane-impermeable dyes might be active in some vacuole membranes. External Cl(-), but not Na(+), was required for vacuole formation. DPC suppressed the vacuole formation and increased cell height, and further incubation with DPC increased the number of dead cells. Bumetanide, dimethylamiloride, and HgCl(2) did not suppress the hypotonic stress-induced formation of water vacuoles. These findings suggest that 3T3-L1 cells regulate the intracellular water content through the DPC-sensitive external Cl(-)-dependent vacuole formation during long-term osmotic stress.

Introduction: Triple negative breast cancers (TNBC) are clinically aggressive tumors that lack the expression of ER, PR and HER2 receptors, and therefore do not respond effectively to the available target therapies. The study of the molecular alterations that are present in these tumors can lead to the identification of potential therapeutic targets that can improve patient's outcome. MicroRNAs (miRNAs) are short non-coding sequences that play a role in breast tumorigenesis regulating the expression of cancer-associated genes. In the present study, we investigated miRNAs expression profile in TNBC and non-TNBC tumors and integrated the data with DNA copy number changes (array-CGH) obtained from the same samples, to obtain the most relevant miRNA targets that may be involved in the pathogenesis of TNBC. Methods: Formalin-fixed paraffin-embedded samples from 43 TNBC and 16 non-TNBC cases were profiled for miRNA using the Nanostring system. Significant differentially expressed miRNAs (with at least 2 fold differences and p≤0.05) between the lesions were calculated by the comparative Ct method (ΔΔCt). DNA copy number analysis from the same samples was performed using an Agilent array-CGH platform. The miRNA data was directly integrated with the array-CGH data from the same cases. Combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems (Ingenuity Pathway, Pathway Studio) were performed to identify predicted target functions. Results: The miRNA profiling revealed 89 miRNAs significant differentially expressed between the TNBC and non-TNBC lesions. Using miRBase and MiRDB target prediction databases we identified 3,378 target genes of upregulated miRNAs and 823 for downregulated miRNAs. A number of 15 miRNAs (out of the 89) presented concomintant genomic gains or losses and miRNA increase or decrease expression, respectively. This direct integration of the data reduced the number of miRNA targets to 1,242. Ingenuity pathway analysis on these predicted targets function, identified canonical pathways, including p53, IL-8 and Rac signaling. Gene expression profiling analysis is underway to confirm the regulatory effect of the selected miRNAs on the targets identified. Conclusions: We have observed significant differentially expressed miRNAs in the analysis of TNBC cases in comparison with non-TNBC cases. The integration analysis with DNA copy number data let us to select the miRNAs with concomitant changes in copy number and gene expression and predicted target algorithms identified key pathways that may be related to the TNBC phenotype. The functional validation of these targets, by in vitro and in vivo experimental models, through the assessment of miRNA-mRNA interaction and/or modulation of miRNA expression will allow us to identify the most relevant miRNAs that are mechanistically involved in the pathogenesis of TNBC. Note: This abstract was not presented at the meeting. Citation Format: Mandeep Gill, Bruna Sugita, Silma R. Pereira, Catalin Marian, Xi Li, Yuriy Gusev, Enilze MSF Ribeiro, Iglenir J. Cavalli, Luciane R. Cavalli. Identification of microRNA targets in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1545. doi:10.1158/1538-7445.AM2014-1545

Molecules of the major histocompatibility complex (MHC) bind protein-derived peptide antigens and present them to T cells. This has been a central dogma in modern immunology, and our appreciation of a variety of cell-mediated immune responses has been based only on this paradigm. However, we now know that T cell recognition also involves non-peptide antigens. Studies over the past several years have established a new paradigm that non-MHC-encoded molecules of the CD1 family mediate presentation of lipid antigens to T cells, and unraveled their significant role in microbial immunity, tumor immunology, and autoimmunity. Identification of a novel pathway for T cell activation mediated by CD1 molecules opens a possibility for new therapeutic strategies, including development of lipid-based vaccines.

Background Evidence suggests that older patients are under investigated and undertreated and experience worse outcomes than younger patients (1). Clinicians are unsure about offering chemotherapy to older patients because of uncertainty about whether the likely benefits in terms of survival and QOL are sufficiently worthwhile to an older person when balanced against likely toxicity and costs of treatment. Methods A questionnaire has been developed, piloted and psychometrically validated to try and identify social attitudes towards cancer treatment for the older patient. 1000 questionnaires containing 24 individual and multidimensional questions were sent to a random sample of the general public. Results The response rate, 17%, was low but analyses demonstrated 1). the reliability of the questionnaire 2). clear attitudes to the use of chemotherapy with respect to potential benefit and 3). that respondents understood the complex multivariable treatment scenarios. Although age modified response, QOL and survival benefits remained the overriding consideration. Over 95% of respondents felt that chemotherapy should be considered for patients aged 56 or less but 71.6% felt that it should still be considered even at age 79. 74.6% considered that a survival benefit of 6 months or more, as seen with palliative colorectal or breast cancer, was worthwhile. Survival benefits of between 1 and 10% (as seen with many adjuvant therapies) produced a wide range of responses suggesting that clinicians cannot assume how useful an individual may perceive a treatment. It is now our intention to send the questionnaire to a random sample of health professionals to compare and contrast attitudes.

The effect of alteration of 5' and 3' flanking sequences on the transcription of plant tRNA genes was analysed using an RNA polymerase III-dependent in vitro transcription system derived from nuclei of cultured tobacco cells. A TATA-like sequence and the CAA motif frequently observed upstream of plant tRNA genes, and the poly(T) stretch usually present downstream, were shown to be necessary for efficient re-initiation of transcription. The CAA motif was shown to be a transcription initiation site. Introduction of the CAA and TATA-like motifs into a gene naturally lacking them greatly enhanced transcription by promoting efficient re-initiation.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.

Intracarotid infusion of bradykinin selectively increases the delivery of compounds into brain tumors. This study sought to determine the role of cyclic GMP in increased permeability across the blood-tumor barrier (BTB) after infusion of bradykinin. In permeability studies, 186 Wistar rats with RG2 gliomas and C6 gliomas were used. Transport across the BTB was quantified by autoradiography and reported as a unidirectional transport, Ki, for [14C]dextran (Mr 70,000) and [14C]aminoisobutyric acid (Mr 103,000), with or without inhibition of cyclic GMP-specific phosphodiesterase or soluble guanylate cyclase. We also determined cyclic GMP levels in tumors and normal brain, with or without intracarotid bradykinin infusion, using RIA. Intracarotid infusion of bradykinin selectively increased permeability in RG2 tumors and C6 tumors for both tracers. Simultaneous infusion of bradykinin and a cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (20 mg/kg), resulted in significantly increased permeability across the BTB, compared to intracarotid bradykinin infusion alone. Zaprinast also significantly prolonged the permeability effects of bradykinin. Pretreatment using i.v. infusion of the soluble guanylate cyclase inhibitor, LY-83583 (125 microg/kg), significantly attenuated the bradykinin effect of opening the BTB. Cyclic GMP levels in RG2 and C6 tumors were significantly increased after intracarotid bradykinin infusion (2.8- and 2.2-fold, respectively). Cyclic GMP levels in normal brain were not increased by bradykinin infusion. These results show that increasing cyclic GMP in tumor microvessels can increase permeability in response to bradykinin.

AIMS: To investigate the lack of equilibrium in the regulatory mechanism of the immune system in Behcet's disease (BD), the expression of Fas antigen, an apoptosis related antigen, on peripheral blood lymphocytes from BD patients was analysed. METHODS: Twenty one BD patients were the subjects in this study. Ten healthy adults were examined as controls. Cell surface antigens of lymphocytes were analysed with flow cytometry. RESULTS: There was a significant (p < 0.01) difference in the proportion of CD4 positive cells with CD25 between BD patients with active uveoretinitis (27.6% (SD 8.4%)) and the controls (14.7% (2.3%)), but no significant difference in the proportion of CD4 or CD45RO positive cells with Fas. On the other hand, the proportion of CD8 positive cells with Fas was significantly (p < 0.01) higher in BD patients with active uveoretinitis (45.6% (11.6%)) than in those with inactive uveoretinitis (23.8% (8.1%)) or in the controls (24.4% (2.5%)). The proportion of CD19 positive cells with Fas was also significantly (p < 0.01) higher in BD patients with active uveoretinitis (13.0% (5.0%)) than in the controls (5.1% (2.1%)). CONCLUSION: The insufficient expression of Fas on activated CD4 positive T cells and its high expression on CD8 positive T cells seem to play an important role in the chronic inflammation in BD.

Sometimes a specific treatment is effective in one subgroup but not in another. An indicator allowing quantitative comparison of treatment effect in two subgroups would be useful in clinical medicine. We have developed such an indicator. It is obtained by calculations using Cox's proportional hazard or logistic model with therapy, subgroup, and confounding explanatory variables. The parameter of the interaction between therapy and subgroup can be estimated and tested statistically. The exponential value of the interaction parameter is what we tentatively call the "hazard ratio ratio", meaning the ratio between the treatment effects in two subgroups. The 95% confidence interval of the indicator can also be calculated. As a numerical example, the hazard ratio between the survival times of postoperative gastric cancer patients treated by adjuvant immunochemotherapy and patients without adjuvant immunochemotherapy in a subgroup with high serum glycosidically bound sialic acid (SA) level was lower than that in a low-SA subgroup using an estimate for hazard ratio ratio of less than 0.5 with statistical significance. We propose this indicator be used as a "responder/non-responder ratio" of therapy effect.

To examine the role of tumor necrosis factor-alpha (TNF) in the induction of experimental autoimmune uveoretinitis (EAU), the authors compared in vivo TNF production in EAU-susceptible and EAU-resistant strains of congenic mice and attempted to determine whether TNF can enhance the inflammation in EAU by injection of TNF at the time of immunization.The production of TNF after stimulation with lipopolysaccharide (LPS) in B10.A and B10.D2 mice was measured by bioassay with L929 cells. The incidence and severity of EAU was compared between the group immunized with conventional methods and the group that alternatively received additional subcutaneous injection of recombinant human TNF (rhTNF) at the time of immunization in both B10.A and B10.D2 mice.Serum concentration of TNF after stimulation with 50 micrograms of LPS was significantly higher in B10.A mice than in B10.D2 mice. The incidence of EAU in B10.A mice was 60%, but it was only 10% in B10.D2 mice using the conventional method. Extremely severe chorioretinitis and iridocyclitis occurred in B10.A mice with the injection of rhTNF at the time of immunization for EAU. The incidence of EAU in B10.A and B10.D2 rose to 100% and 40%, respectively. When administered alone, rhTNF did not cause any inflammatory change in the uvea.The rhTNF was found to enhance the immune response to interphotoreceptor retinoid-binding protein in mice. These results suggest that susceptibility to EAU is in some part mediated by the ability of mice to produce TNF.

A novel series of glycosphingolipids containing choline phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar glycolipids revealed the presence of more than three components with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragendorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sephadex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound choline phosphate. The structural elucidation of the oligosaccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and methylation analysis. Thus, the structures of PGL1 and PGL2 were deduced to be as follows: cholinephosphoryl--6Gal beta 1-1Cer and cholinephosphoryl--6Gal beta 1-6Gal beta 1-1Cer. Although the oligosaccharide structures of both PGL1 and PGL2 have previously been found in other organisms, the presence of a choline phosphate group as an oligosaccharide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecasphingenines and their nonadecasphingenine homologues.