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In this study, eight naphthoquinone derivatives were synthesized in yields ranging from 52 to 96% using easy, fast, and low-cost methodologies. All naphthoquinone derivatives were screened for their in vitro anti-proliferative activities against OVCA A2780 cancer cell line. Amongst all analysed compounds, derivatives 3 - 5 presented the most prominent cytotoxic potential. Naphthoquinones 3 and 4, bearing sulfur-containing groups, were identified as having high potential for ROS production, in particular the superoxide anion. Furthermore, 3 and 4 compounds caused a decrease in the cell population in G0/G1 and induced more than 90% of the cell population to apoptosis. Compound 5 did not act in any of these processes. Finally, compounds 3 - 5 were tested for their inhibitory ability against PI3K and MAPK. Compounds 3 and 4 do not inhibit the PI3K enzyme. On the other hand, the naphthoquinone-polyphenol 5 was only able to inhibit reduced the percentage of cells expressing pERK.

Genome wide-association studies (GWAS) have established over 400 breast cancer risk loci defined by common single nucleotide polymorphisms (SNPs), including several associated with estrogen-receptor (ER)-negative disease. Most of these loci have not been studied systematically and the mechanistic underpinnings of risk are largely unknown. Here we explored the landscape of genomic features at an ER-negative breast cancer susceptibility locus at chromosome 2p23.2 and assessed the functionality of 81 SNPs with strong evidence of association from previous fine mapping. Five candidate regulatory regions containing risk-associated SNPs were identified. Regulatory Region 1 in the first intron of WDR43 contains SNP rs4407214, which showed allele-specific interaction with the transcription factor USF1 in in vitro assays. CRISPR-mediated disruption of Regulatory Region 1 led to expression changes in the neighboring PLB1 gene, suggesting that the region acts as a distal enhancer. Regulatory Regions 2, 4, and 5 did not provide sufficient evidence for functionality in in silico and experimental analyses. Two SNPs (rs11680458 and rs1131880) in Regulatory Region 3, mapping to the seed region for miRNA-recognition sites in the 3' untranslated region of WDR43, showed allele-specific effects of ectopic expression of miR-376 on WDR43 expression levels. Taken together, our data suggest that risk of ER-negative breast cancer associated with the 2p23.2 locus is likely driven by a combinatorial effect on the regulation of WDR43 and PLB1.

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.

Osteopontin (OPN) is upregulated in several types of tumor and has been associated with chemoresistance. However, the contribution of OPN splicing isoforms (OPN‑SIs) to chemoresistance requires further investigation. The present study aimed to evaluate the expression patterns of each tested OPN‑SI in cisplatin (CDDP)‑resistant ovarian carcinoma cell lines, focusing on the role of the OPN‑c isoform (OPNc) in drug resistance. ACRP ovarian cancer cells resistant to CDDP, as well as their parental cell line A2780, were used. Analyses of the transcriptional expression of OPN‑SIs, epithelial‑mesenchymal transition (EMT) markers and EMT‑related cytokines were performed using reverse transcription‑quantitative PCR. OPNc was silenced in ACRP cells using anti‑OPNc DNA oligomers and stably overexpressed by transfecting A2780 cells with a mammalian expression vector containing the full length OPNc cDNA. Functional assays were performed to determine cell proliferation, viability and colony formation. The results demonstrated that among the three tested OPN‑SIs, OPNc was the most upregulated transcript in the ACRP cells compared with the parental A2780 cells. In addition, the expression levels of P‑glycoprotein multidrug transporter were upregulated in CDDP‑resistant ACRP cells compared with those in A2780 cells. OPNc knockdown sensitized ACRP cells to CDDP treatment and downregulated P‑gp expression levels compared with those in the negative control group. Additionally, silencing of OPNc impaired cell proliferative and colony formation abilities, as well as reversed the expression levels of EMT markers and EMT‑related cytokines compared with those in the negative control cells. Notably, although stable OPNc overexpression resulted in increased A2780 cell proliferation, it notably increased CDDP sensitivity compared with that in the cells transfected with a control vector. These results suggested that OPNc silencing may represent a putative approach to sensitize resistant ovarian cancer cells to chemotherapeutic agents.

Purpose BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification. Methods We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification. Results Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach. Conclusion High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

To date, genome-wide association studies (GWASs) have identified 39 genomic loci associated with risk of epithelial ovarian cancer at genome-wide significance level (p ≤ 5 × 10−8) and 13 additional loci using less strict thresholds. Follow-up functional dissection of these loci to uncover the underlining mechanisms driving cancer susceptibility has been challenging. In a manner similar to how post-linkage studies led the characterization of then poorly understood cellular pathways, functional analysis of GWAS loci is revealing new mechanisms of ovarian cancer. Here, we review recent methodological and conceptual progress relevant to the understanding of how common genetic variation influences the risk of epithelial ovarian cancer.

Background Nucleus accumbens-associated protein 1 (NAC1) has multifaceted roles in cancer pathogenesis and progression, including the development of drug resistance, promotion of cytokinesis, and maintenance of "stem cell-like" phenotypes. NAC1 is a transcriptional co-regulator belonging to the bric-a-brac tramtrack broad (BTB) family of proteins, although it lacks the characteristic DNA binding motif of the BTB family. The formation of higher-order transcription complexes likely depends on its interaction with other DNA-binding co-factors. Results NAC1 interacts with BCL6 via its C-terminal BEN domain and forms a complex that binds the promoter region and activates transcription of the NAC1 target gene, FOXQ1. NAC1 and BCL6 were coordinately upregulated. Our analysis also identified a novel function of NAC1 in attenuating BCL6 auto-downregulation in ovarian cancer. Lastly, we found a significant overlap among NAC1- and BCL6-regulated genes in tumor cells, suggesting that NAC1 and BCL6 coordinately control transcription in cancer. Conclusions The results of this study provide a novel mechanistic insight into the oncogenic roles of NAC1 and underline the importance of developing the NAC1/BCL6-targeted cancer therapy. Methods Using the Cistrome database and Chromatin Immunoprecipitation (ChIP) analyses, we identified BCL6 as a potential NAC1- interacting molecule. Co-immunoprecipitation (Co-IP), luciferase reporter assay, immunohistochemistry and microarray analysis were performed to analyze the interaction between NAC1 and BCL6 and the mechanisms by which they regulate the downstream genes including FOXQ1.

Background Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Primary cytoreductive surgery with adjuvant taxane-platinum chemotherapy is the standard treatment to fight ovarian cancer, however, their side effects are severe, and chemoresistance emerges at high rates. Therefore, EOC clinic urges for novel treatment strategies to reverse chemoresistance and to improve the survival rates. Metformin has been shown to act in synergy with certain anti-cancer agents, overcoming chemoresistance in various types of tumors. This paper aims to investigate the use of metformin as a new treatment option for cisplatin- and paclitaxel-resistant ovarian cancer. Methods The effects of metformin alone or in combination with conventional drugs on resistant EOC cell lines were investigated using the MTT assay for cell proliferation; Flow Cytometry analysis for cell cycle and the mRNA expression was analyzed using the real-time PCR technique. Results We found that metformin exhibited antiproliferative effects in paclitaxel-resistant A2780-PR, and in cisplatin-resistant ACRP cell lines. The combined therapy containing conventional drugs and metformin improved the effect of the treatment in cell proliferation rate, especially in the resistant cells. We found that metformin, in clinical relevant doses, could significantly reduce the mRNA expression of inflammatory cytokines and NF-κB signaling pathway. Conclusions Taken together, our observations suggest that metformin inhibits the inflammatory pathway induced by paclitaxel and cisplatin treatment. Furthermore, metformin in combination with paclitaxel or cisplatin improved the sensitivity in drug-resistant ovarian cancer cells. Therefore, metformin may be beneficial treatment strategy, particularly in patients with tumors refractory to platinum and taxanes.

Background/Aims: Osteoporosis is a bone metabolic disease that affects mostly post-menopausal women. There has been shown that vitamin K (VK) supplementation during menopause may decrease bone loss as well as risk of bone breaking. Aiming to clarify the beneficial role of VK in bone metabolism during menopause, we investigated mineral metabolism and bone ultrastructure of ovariectomized (OVX) mice. Methods: To determine the effects chronic use of VK in bone structure and mineral metabolism in OVX mice, we used several methods, such as DXA, µCTScan, and SEM as well as biomolecular techniques, such as ELISA and qRT-PCR. In addition, complete analysis of serum hormonal and other molecules associated to bone and lipid metabolism were evaluated overview the effects of VK in menopause murine model. Results: VK treatment significantly affects Pi metabolism independently of OVX, changing Pi plasma, urinary output, balance, and Pi bone mass. Interestingly, VK also increased VLDL in mice independently of castration. In addition, VK increased compact bone mass in OVX mice when we evaluated it by DXA, histomorphometry, µCTScanning. VK increased bone formation markers, osteocalcin, HYP- osteocalcin, and AP whereas it decreased bone resorption markers, such as urinary DPD/creatinine ratio and plasmatic TRAP. Surprisingly, SEM images revealed that VK treatment led to amelioration of microfractures observed in OVX untreated controls. In addition, SHAM operated VK treated mice exhibited higher number of migrating osteoblasts and in situ secretion of AP. OVX led to decreased to in situ secretion of AP that was restored by VK treatment. Moreover, VK treatment increased mRNA expression of bone Calbindin 28KDa independently of OVX. Conclusion: VK treatment in OVX mice exhibited beneficial effects on bone ultrastructure, mostly by altering osteoblastic function and secretion of organic bone matrix. Therefore, VK could be useful to treat osteopenic/osteoporotic patients.

Background/aims Tributyltin (TBT) is an organotin (OTs) and biohazard organometallic pollutant. Recently our group has shown that TBT, even in very low doses, has deleterious effects on several tissues most likely due to its role as an endocrine-disrupting molecule. Other studies have confirmed that OT exposure could be responsible for neural, endocrine, and reproductive dysfunctions via in vitro and in vivo models. However, TBT effects on bone lack concise data despite the fact that bone turnover is regulated by endocrine molecules, such as parathormone (PTH), estrogen (E2), etc. Our group has already shown that TBT disrupts adrenal and female gonadal functions. Methods We studied the effects of TBT on bone metabolism and structure using DXA, microCT scan, and SEM. We also determined the calcium (Ca²⁺) and phosphate (Pi) metabolism in TBT-treated rats as well as some biomarkers for bone formation and resorption. Results Surprisingly, we found that TBT leads to higher bone mineral density (BMD) although lesions in spinal bone were observed by either microCT scan or SEM. Biomarkers for bone resorption, such as the urinary deoxipyridinolines (DPD) excretion ratio was increased in TBT-treated animals versus mock-treated controls. Osteocalcin (OC) and alkaline phosphatase (AP) are markers of bone formation and are also elevated suggesting that the bone matrix suffers from a higher turnover. Serum Ca²⁺ (total and ionized) do not changed by TBT treatment although hypercalciuria is observed. Conclusion It is known that Sn atoms have three valence states (Sn²⁺, Sn³⁺, and Sn⁴⁺); hence, we hypothesized that Sn (more likely Sn²⁺) could be competing with Ca²⁺ and/or Mg²⁺ in hydroxyapatite mineral matrix to disturb bone turnover. Further work is needed to confirm this hypothesis.

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Indeed, EOC epidemiological scenario is partially due to its late diagnosis, when most of tumor cells have acquired metastatic and chemoresistant phenotype. Despite the initial satisfactory response of EOC cells to platinum/taxanes-based therapy, chemoresistance emerges in an uncontrolled pace, specifically when the patient carries a high-grade serous adenocarcinoma (HGS-AC). Therefore, EOC clinic urges for novel treatment possibilities, aiming the reversal of chemoresistance, the improvement of patients’ quality of life, and their survival rates. cDNA microarray data from our group demonstrated that chemokines secreted to the tumor microenvironment, as CXCL2, IL-6, and IL-8 likely play a crucial role in EOC cells chemoresistance. Thereafter, the present study focused on the role of CXCL2, which binds to its receptor CXCR2 in the membrane of target cells. Nonetheless its function in HGS-AC is unclear. Our study model was based in two HGS-AC lineages: chemosensitive A2780, and its pan-resistant derived ACRP (IC50 to cisplatin 7.3uM vs. 26,56uM, p Citation Format: Taciane Henriques, Diandra dos Santos, Isabella Guimaraes, Nayara Tessarollo, Paulo Lyra, Patricia Mesquisa, Diana Padua, Ana Luisa Amaral, Luisa Pereira, Bruno Cavadas, Ian Silva, Raquel Almeida, Leticia Rangel. Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A133. doi:10.1158/1535-7163.TARG-19-A133

Ethnic differences in patient genetics and breast cancer (BC) biology contribute to ethnic disparities in cancer presentation and patient outcome. We prospectively evaluated SNPs within phase I and phase II tamoxifen (TAM) metabolizing enzymes, and the estrogen receptor gene (ESR1), aiming to identify potential pharmacogenomic ethnicity patterns in an ER-positive BC cohort constituted of Hispanic and Non-Hispanic White (NHW) women in South Texas. Plasma concentrations of TAM/metabolites were measured using HPLC. CYP2C9, CYP2D6 and SULT1A1 genotypes were determined by DNA sequencing/Pyrosequencing technology. ESR1 PvuII and XbaI SNPs were genotyped using Applied Biosystems Taqman® Allelic Discrimination Assay. Hispanics had higher levels of TAM, 4-hydroxytamoxifen, and endoxifen than NHWs. There was a higher prevalence of CYP2D6 EM within Hispanics than NHWs, which corresponded to higher endoxifen levels, but no differences were verified with regard to CYP2C9 and SULT1A1. We found a higher incidence of the wild type forms of the ESR1 in Hispanics than NHWs. The performance status, the disease stage at diagnosis, and the use of aromatase inhibitors might have overcome the overall favorable pharmacogenomics profile of Hispanics when compared to NHWs in relation to TAM therapy responsiveness. Our data strongly point to ethnical peculiarities related to pharmacogenomics and demographic features of TAM treated Hispanics and NHWs. In the era of pharmacogenomics and its ultimate goal of individualized, efficacious and safe therapy, cancer studies focused on the Hispanic population are warranted because this is the fastest growing major demographic group, and an understudied segment in the U.S.

Cancer is one of the leading causes of death worldwide. Every year 8.2 million people die from the disease. In this context, breast and ovarian cancer are the most incidental among women. Elucidation of cell growth pathways and the observation that these pathways are altered in human cancer have encouraged the search for specific inhibitors. The phosphatidylinositol-3cinase (PI3K)/Protein kinase b (AKT)/Mammalian Target of Rapamycin (mTOR) is an important pathway involved in cell growth, tumorigenesis, cell invasion, and resistance to therapies. This pathway is often activated in breast and ovarian cancers and the deregulation of its signaling can contribute to tumor growth, angiogenesis and metastasis. Metformin is one of the most commonly prescribed antidiabetic drugs in the world whose anticancer effects, mediated by reduced mTOR signaling, have become notable. Therefore, this review provides an overview of signaling pathway PI3K/AKT/mTOR in the ovarian and breast cancers as well as for target therapies of mTOR signaling, with an emphasis on its mechanisms, clinical applicability and future perspectives.

Estrogen is a steroidal hormone involved in several physiological functions in the female body including regulation of serum lipid metabolism and breast cancer (BC). Estrogen actions on serum lipids mostly occur through its binding to intracellular Estrogen Receptor alpha (ERalpha) isoform, expressed in most of tissues. This gene (ESR1) exhibit many polymorphic sites (SNPs) located either on translated and non-translated regions that regulate ERalpha protein expression and function. This paper aimed to investigate the association of two intronic SNPs of ESR1 gene, namely c454-397T>C (PvuII) and c454-351A>G (XbaI) to alterations in serum levels of total cholesterol (T-chol), total lipid (TL), low density lipoprotein cholesterol (LDL), high density lipoprotein (HDL), and triglycerides (TG) in a cohort of post-menopausal women. In addition, we aimed to associate presence of these SNPs to development of BC along 5 years period. To do so, a group of healthy 499, highly miscigenated, post-menopausal Brazilian women, were carried using PCR-FRLP technique and further confirmed by automatic sequence analysis as well followed through 5 years for BC incidence. Measurements of serum lipid profile by standard commercial methods were carried individually whereas Dual Energy X-ray Absorciometry (DXA) measured Body Mass Indexes (BMI), Fat Mass (FM), Lean Body Mass (LBM), and Body Water Content (BWC). No effects of PvuII SNP on ESR1 gene were observed on patient´s serum T-chol, TL, LDL, HDL, and TG. However, c454-397T>C PvuII SNP is associated to lower body fat and higher levels of lean mass and body water and lower incidence of BC. On the other hand, statistically significant effect of XbaI c454-351A>G SNP on serum TG and TL levels. Patients homozygous for X allele were followed up from 2010–2015. They showed higher incidence of breast cancer (BC) when compared to either heterozygous and any P allele combination. Moreover, the increasing of TG and TL serum concentrations associated to SNP XbaI c454-351A>G on ESR1 gene is enhanced in both obese (higher BMI) and elder women. Taken together, these results suggested that XbaI c454-351A>G SNP is associated to changes in lipid profile, particularly in serum TG and TL, in this cohort of post-menopausal woman. Also, this paper shows another link between obesity and BC corroborating the current thesis that both diseases are interlinked.

Osteoporosis is one of the major diseases that affects mostly postmenopausal women. Despite being a multifactorial disease, some genes have been shown to play an important role in osteoporosis. Bone mineral density (BMD) is still largely used to diagnose it, although many other biomarkers are used to better follow the disease onset. It has been shown that the apolipoprotein E (APOE) gene could be a biomarker for risk of fractures as well as to predict lower BMD in patients with osteoporosis. The human APOE gene encodes 3 protein isoforms called ApoE2, ApoE3, and ApoE4, resulting in 4 possible genotypes, because they are a product of a single nucleotide polymorphism found in this gene. So far, the APOE4 allele has been associated with low BMD in postmenopausal women and to incidence of bone breaking in older women. This study aimed to investigate the role of ApoE isoforms in a cohort of 413 postmenopausal Brazilian women. These patients were randomly recruited, clinically examined, and subjected to dual-energy X-ray absorptiometry to measure their BMD. Patients were further grouped as normal BMD (T-score 0.5) or low BMD (T-score 1.0, osteopenic or osteoporotic). Patients with osteopenia or osteoporosis were further genotyped for APOE alleles as well as tested for many serum bone turnover biomarkers. Our data showed that presence of the APOE3 allele was associated with both higher BMDs and higher serum concentrations of osteocalcin and alkaline phosphatase, biomarkers for bone formation. On the other hand, the APOE2 and APOE4 alleles were associated with lower BMD as well as higher levels of serum C-terminus collagen peptide and urinary deoxipyridinolines, biomarkers for bone resorption. However, these effects on lower BMD and bone resorption biomarkers observed in either APOE2 or APOE4 alleles were eliminated when patients' genotype carried the APOE3 allele. Codominance of the APOE3 allele was also associated with lesser cases of bone fractures in these patients within a 5-year follow-up. In conclusion, our data show that APOE4 may be associated with lower bone formation as well as increased risk of osteoporosis and bone fractures, whereas APOE3 seems to decrease lowering BMD in postmenopausal women, and its presence seemed to lower the incidence of bone breaking in patients with osteoporosis.

Breast cancer (BC) is the second most common kind of cancer in the world and the most common among women. Its frequency rate is becoming alarming, being this way a major challenge to global health. It is worth mentioning that the lack of effective therapeutic options, in particular for certain subtypes, such as triple-negative tumor, still present a challenge for clinicians who often have to resort to highly unspecific cytotoxic therapies. Unfortunately, most patients relapse after a period of remission and eventually tumors becomes refractory to frontline therapy. This makes it desirable to identify drugs which synergize with chemotherapeutics and potentially reduce the dose of them that is necessary to treat patients. In the current financial and economical context, drug repositioning is gaining increasing interest as an alternative strategy for drug development. Cancer cells frequently exhibit specific alterations in their metabolic activity. Thus, targeting lipid metabolism in cancer cells could have therapeutic benefits and it does appear as promising auxiliary strategies in the fight against cancer. The present work aimed to investigate the effect of atorvastatin (ATV) on paclitaxel (PTX), cisplatin (CDDP) and olaparib (OLAP) cytotoxic on human BC cell lines MDA-MB-231 and MCF-7, and the mechanisms by which this interaction occurs. ATV decreased cellular metabolic viability (CMV; MTT assay) in a dose-time-dependent manner, especially in MDA-MB-231 being the maximum effect observed in with ATV 100µM (- 90%; p Note: This abstract was not presented at the meeting. Citation Format: Diandra Zipinotti dos Santos, Isabella dos Santos Guimarães, Nayara Gusmão Tessarollo, Taciane Barbosa Henriques Barbosa Henriques, Paulo Cilas Lyra Junior, Marcele L L. de Souza, Maria C. Gomes, Ian Victor Silva, Leticia B. A. Rangel. Enhancement of cisplatin activity against triple negative breast cancer cells by atorvastatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5276.

OBJECTIVE: To test the effectiveness of combining conventional antineoplastic drugs (cisplatin and etoposide) with metformin in the treatment of non-small cell lung cancer in the NCI-H460 cell line, in order to develop new therapeutic options with high efficacy and low toxicity.METHODS: We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and calculated the combination index for the drugs studied.RESULTS: We found that the use of metformin as monotherapy reduced the metabolic viability of the cell line studied. Combining metformin with cisplatin or etoposide produced a synergistic effect and was more effective than was the use of cisplatin or etoposide as monotherapy.CONCLUSIONS: Metformin, due to its independent effects on liver kinase B1, had antiproliferative effects on the NCI-H460 cell line. When metformin was combined with cisplatin or etoposide, the cell death rate was even higher.

Objectives: In this study we present the development of a database of psychoactive drugs dispensed to patients attended by the Brazilian Public Health System (SUS) in the city of Manhuacu, Minas Gerais and the pattern of drug prescription in this city. Methods: 827 patients under psychoactive treatment and attended by SUS were surveyed and information such as gender, degree of education, age, marital status were collected. The collected data were analyzed in order to outline patients' profile and the dispensing and information was used to the access the pattern of psychoactive drug use in the city. Results: Women accounted for 67.2% of the population and age seemed to influence positively the use of psychoactive drugs. Benzodiazepines and antidepressants were among the most prescribed drugs especially after 20 years of age, while in the younger population the antipsychotics and antiepileptics were the mainly prescribed drugs. Antiepileptics/mood stabilizers seemed to be prescribed mainly to single men and women. Conclusion: Personal data concerning gender, age and marital status are related with psychoactive drug dispensing. The collected data will serve as a support for the performance of pharmacists responsible for dispensing psychoactive drugs in the municipality.

Objective : To evaluate the association of −397T>C and −351A>G single nucleotide polymorphisms (SNPs) – also called PvuII and XbaI, respectively – located on estrogen receptor alpha (ERS1) gene with age at menarche, menopause onset, fertility and miscarriage in a population of post-menopausal women. Study design : Cross-sectional study with 273 healthy, high miscegenated, post-menopausal women (mean age of 63.1 ± 9.7 years old). Subjects were genotyped for PvuII and XbaI SNPs by PCR-RFLP and confirmed by automatic sequencing. Reproduction informations (age at menarche, age at menopause, number of pregnancies, fertility rate and miscarriages) were obtained by retrospective study using a questionnaire. Result(s) : Age at menarche, menopause onset, number of pregnancies, total fertility rate, and parity did not seem to be influenced by any of the studied genotypes (chi-square, p > 0.05). However, women carrying the xx genotype showed a 44% higher chance of miscarriage, whereas this value did not trespass 16% for any other genotype analyzed. It has been also observed a higher occurrence of miscarriage in association with combined xxpp genotype of ERS1 gene (chi-square, p Conclusion(s) : The present data indicate that the studied SNPs on ERS1 gene do not influence the menstrual cycle timing and parity but there is a strong relationship between the xx ERS1 SNP genotype and the incidence of miscarriage in the post-menopausal population analyzed.

O estrogênio é um hormônio esteroide sexual que possui efeito sobre o sistema reprodutor feminino e masculino. Esses efeitos são mediados principalmente pelas isoformas α e ß (ER α e ß), componente da superfamília de receptores nucleares que controlam a transcrição gênica. De fato, a resposta do gene ao estrogênio depende de muitos fatores, incluindo a avaliação dos subtipos de ER, os correguladores, o tempo de exposição ao estrogênio e a quantidade desse hormônio. Os processamentos (do inglês splicing) alternativos geram diversas variantes de RNAm de ESR1. As isoformas de RNAm com distintas regiões 5' não traduzidas resultam na expressão da proteína ESR1 de diferentes tamanhos. Sabe-se que o gene ESR1 possui muitos sítios de polimorfismos que podem ser responsáveis por diferentes variantes alélicas da proteína, podendo alterar a função e a atividade dessa proteína e, então, resultar nas diferenças do efeito do estrogênio sobre o desenvolvimento de doenças. Existem vários fatores de risco relacionados ao BRCA, porém os polimorfismos do gene ESR1 contribuem de maneira expressiva para carcinogênese mamária. Os SNPs Pvu II e Xba I e os STRs (GT)n e (TA)n despertam curiosidade por se localizarem em regiões não traduzidas do gene ESR1 e poderem estar relacionados a doenças de grande impacto, como o BRCA. Nota-se certa interferência desses polimorfismos nessa neoplasia, porém os resultados são divergentes. Contudo, é importante ampliar o conhecimento da genética do ESR1, pois existem evidências que suas propriedades interferem no desenvolvimento do BRCA.

Eleven 2,3-(substituted)-1,4-naphthoquinone derivatives were synthesized in yields ranging from 52-89%. These derivatives were evaluated for their cytotoxic effects on human lungs (H460), triple-negative breast (MDA-MB-231) and ovarian (A2780) cancer cell lines. Compounds 5f and 8 showed IC50 values of 3.048 × 10-5 mol L-1 and 4.24 × 10-6 mol L-1 for H460; 5c and 8 showed IC50 values of 2.16 × 10-5 mol L-1 and 1.60 × 10-5 mol L-1 for MDA-MB-231, and 5g and 8 showed IC50 values of 2.68 × 10-6 mol L-1 and 3.89 × 10-6 mol L-1 for A2780. Additionally, we conducted a docking study with the four most active compounds and the therapeutic targets PI3K and topoisomerase II showing the pharmacophoric conformation of these compounds.

In previous papers we showed that Ang II increases the proximal tubule Na+-ATPase activity through AT1/PKC pathway [L.B. Rangel, C. Caruso-Neves, L.S. Lara, A.G. Lopes, Angiotensin II stimulates renal proximal tubule Na+-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316, L.B.A. Rangel, A.G. Lopes, L.S. Lara, C. Caruso-Neves, Angiotensin II stimulates renal proximal tubule Na+)-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316]. In the present paper, we study the involvement of PI-PLCbeta on the stimulatory effect of angiotensin II (Ang II) on the proximal tubule Na+-ATPase activity. Western blotting assays, using a polyclonal antibody for PI-PLCbeta, show a single band of about 150 KDa, which correspond to PI-PLCbeta isoforms. Ang II induces a rapid decrease in PIP2 levels, a PI-PLCbeta substrate, being the maximal effect observed after 30 s incubation. This effect of Ang II is completely abolished by 5 x 10(-8) M U73122, a specific inhibitor of PI-PLCbeta. In this way, the effect of 10(-8) M Ang II on the proximal tubule basolateral membrane (BLM) Na+-ATPase activity is completely abolished by 5 x 10(-8) M U73122. The increase in diacylglycerol (DAG) concentration, an product of PI-PLCbeta, from 0.1 to 10 nM raises the Na+-ATPase activity from 6.1+/-0.2 to 13.1+/-1.8 nmol Pi mg(-1) min(-1). This effect is similar and non-additive to that observed with Ang II. Furthermore, the stimulatory effect of 10 nM DAG is completely reversed by 10(-8) M calphostin C (Calph C), an inhibitor of PKC. Taken together these data indicate that Ang II stimulates the Na+-ATPase activity of proximal tubule BLM through a PI-PLCbeta/PKC pathway.

A better understanding of changes in gene expression during ovarian tumorigenesis and the identification of specific tumor markers may lead to novel strategies for diagnosis and therapy for this disease. Using our serial analysis of gene expression (SAGE) data, as well as public SAGE databases that contained a total of 137 SAGE libraries representing a wide variety of normal and neoplastic tissues, we identified five novel SAGE tags specifically expressed in ovarian cancer. Database analysis, cloning and, sequencing of the corresponding expressed sequence tags revealed details about these transcripts that we named human ovarian cancer-specific transcripts (HOSTs). HOST1 was found to be identical to the gene encoding ovarian marker CA125 (MUC16). HOST2 is a novel gene containing multiple copies of retroviral-related sequences without an obvious open reading frame. HOST3 encodes the tight-junction protein claudin-16 (CLDN16). HOST4 encodes a poorly characterized proteoglycan link protein (LP), and HOST5 codes for a type II sodium-dependent phosphate transporter (SLC34A2). Except for MUC16, these genes have not previously been shown to be expressed in ovarian or other cancers. Northern blot analysis confirmed that HOST genes are rarely expressed in normal tissues or nonovarian cancers, but are frequently expressed in ovarian cancer-derived cell lines and primary tumors. Moreover, HOST genes are upregulated in all four major subtypes of ovarian cancer compared to cultivated ovarian surface epithelial cells, as concluded by real-time reverse transcription (RT)-PCR using a panel of microdissected ovarian tumors. The sodium-dependent phosphate transporter (HOST5/SLC34A2) expression was associated with increased differentiation in ovarian serous tumors. While the roles of HOSTs in ovarian malignant transformation remain unclear, we propose that HOSTs may represent alternative targets for diagnosis and therapy and of this deadly disease.

Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

Background Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines. Results Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ERα positive (ERα+) and ERα negative (ERα−) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ERα + tumors. To further investigate the role of estrogen, we conducted 17β-estradiol (E2) and demethylating agent 5-aza-2′-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer. Conclusions Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0045-9) contains supplementary material, which is available to authorized users.

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.

Angiotensin-(1-7) (Ang-(1-7)) modulates the Na+-ATPase, but not the Na+,K+-ATPase activity present in pig kidney proximal tubules. The Na+-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1-7) (68% by 10(-9) M), in a dose-dependent manner. This effect is due to an increase in Vmax, while the apparent affinity of the enzyme for Na+ is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1-7) effect is mediated by receptor. The Ang-(1-7) stimulatory effect is not changed by either PD 123319, an AT2 receptor antagonist, or A779, an Ang-(1-7) receptor antagonist. On the other hand, increasing the concentration of the AT1 receptor antagonist losartan from 10(-11) to 10(-9) M, reverses the Ang(1-7) stimulation completely. A further increase to 10(-3) M losartan reverses the Na+-ATPase activity to a level similar to that obtained with Ang-(1-7) (10(-9) M) alone. The stimulatory effect of Ang-(1-7) at 10(-9) M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na+-ATPase activity is restored to control values. These data suggest that Ang-(1-7) selectively modulates the Na+-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na+-ATPase activity by angiotensin II.

Angiotensin II (AG II) stimulates the ouabain-insensitive, furosemide- sensitive Na+-ATPase present in the basolateral membrane of pig renal proximal tubules in a dose dependent manner. Maximum effect was obtained with 10-8 M AG II, which corresponded to an activity 134% higher than control. Half of the maximum effect was observed between 10-11 M and 10-10 M, corresponding to physiological hormone levels. Saralasin, an AG II peptide analogue receptor antagonist, abolished the phenomenon, demonstrating that AG II interacts with specific sites in pig proximal tubules. The AG II stimulatory effect was also prevented by dithiothreitol (DTT), a reducing compound, and by 10 nM losartan, a non-peptide antagonist highly specific for AT1 receptors, characterizing AG II binding to AT1 receptors. GTPgammaS, a non-hydrolysable GTP analogue, increased by 159% the enzyme activity as compared to the control values. The simultaneous addition of 10-5 M GTPgammaS and 10-8 M AG II did not have additive effects. Furthermore, the stimulatory action of AG II was completely abolished by 0.1 microM GDPbetaS, a non-hydrolysable GDP analogue. Two microgram ml-1 pertussis toxin, an inhibitor of Gi-protein, did not modulate the AG II stimulatory effect. On the other hand, the Na+-ATPase activity was enhanced 100% in the presence of cholera toxin and 85% in the presence of both AG II and cholera toxin. Taken together, these data suggest that AG II activates the Na+-ATPase activity through AT1 receptors coupled to a pertussis-insensitive and cholera-sensitive G-protein.

Breast cancer (BC) hormonal receptors status is assessed by immunohistochemistry (IHC), a specific, sensitive, and accessible method that guide breast cancer treatment. In this study, we evaluated progesterone receptor (PR) expression in 53 BC cases using 3 anti-PgR antibodies (AB): monoclonal (SP42 and PgR636) and polyclonal ab62621. Primary BC cases (with signed informed consent) were used to generate tissue microarray platforms, where PR expression was accessed by IHC and evaluated by the Allred score. Categorical and quantitative data are shown in percentage and mean, respectively. Concordance (CON) and correlation among ABs were analyzed by Kappa factor (Κ), Spearman’s correlation coefficient (ρ) or intraclass correlation coefficient. Staining patterns of each AB were compared by paired T-Test. We noted poor CON and Κ between ab62621 vs SP42 (CON=64.1%; Κ=0.247), and ab62621 vs PgR636 (CON=62.3%; Κ=0.204), but higher CON between SP42 vs PgR636 (CON 90.6%; Κ=0.738). Data were corroborated by Mc Nemar statistical test (p=0.019, p=0.014 and p>0.05, respectively). Regarding staining intensity (SI) among PgR+ samples, we found higher proportion of weak staining and lower SI for ab62621 (48.3%; mean IS=1.6), when compared to SP42 (20.0%, mean IS=2.1, T-test p

Indium (III) phthalocyanine (InPc) was encapsulated into nanoparticles of PEGylated poly(d,l-lactide-co-glycolide) (PLGA-PEG) to improve the photobiological activity of the photosensitizer. The efficacy of nanoparticles loaded with InPc and their cellular uptake was investigated with MCF-7 breast tumor cells, and compared with the free InPc. The influence of photosensitizer (PS) concentration (1.8–7.5 μmol/L), incubation time (1–2 h), and laser power (10–100 mW) were studied on the photodynamic effect caused by the encapsulated and the free InPc. Nanoparticles with a size distribution ranging from 61 to 243 nm and with InPc entrapment efficiency of 72 ± 6 % were used in the experiments. Only the photodynamic effect of encapsulated InPc was dependent on PS concentration and laser power. The InPc-loaded nanoparticles were more efficient in reducing MCF-7 cell viability than the free PS. For a light dose of 7.5 J/cm2 and laser power of 100 mW, the effectiveness of encapsulated InPc to reduce the viability was 34 ± 3 % while for free InPc was 60 ± 7 %. Confocal microscopy showed that InPc-loaded nanoparticles, as well as free InPc, were found throughout the cytosol. However, the nanoparticle aggregates and the aggregates of free PS were found in the cell periphery and outside of the cell. The nanoparticles aggregates were generated due to the particles concentration used in the experiment because of the small loading of the InPc while the low solubility of InPc caused the formation of aggregates of free PS in the culture medium. The participation of singlet oxygen in the photocytotoxic effect of InPc-loaded nanoparticles was corroborated by electron paramagnetic resonance experiments, and the encapsulation of photosensitizers reduced the photobleaching of InPc.

There are several risk factors related to Breast Cancer (BC) risks and response to chemotherapy with SERMs. Recently some single nucleotide polymorphisms (SNPs) on ESR1 gene have been associated to this disease. However, data are still inconclusive. The present study aimed to investigate the association of SNPs c454-397T>C (also called PvuII) and c454-351A>G (so called XbaI) to incidence of sporadic BC; ERα expression in BC; tamoxifen hormonetherapy (HT-TMX) responsiveness. To do so, a cohort of BC patients was analyzed through retrospective data collection, immunohistochemistry to ERα protein, and genotyping for PvuII and XbaI SNPs by PCR-RFLP, confirmed by sequencing. Significant difference in PvuII alleles frequencies were found BC patients when compared to control samples. Patients with P allele have a 5.14-fold increased BC risk. We found higher P and X alleles frequencies in ERα positive BC and the pp and xx genotypes were observed exclusively in patients with HT-TMX-responsive BC. Taken together, data indicates that P allele as a novel sporadic BC biomarker whereas p and x alleles enhanced chemotherapy responsiveness.

Resistance to chemotherapeutic drugs corresponds to main causes of treatment failure. Total osteopontin (OPN) has been described as a gene product involved on chemoresistance, besides activating several aspects of tumor progression. OPN regulates the expression of P-glycoprotein (P-gp) in prostate cancer (PCa) cells, besides promoting apoptosis evasion induced by chemotherapy using paclitaxel and estramustin. These data point OPN as a potential therapeutic target to approaches that could reduce chemotherapy tumor resistance. However, most of this data are related to total OPN. Once OPN suffers alternative splicing, producing 3 isoforms, further investigation should demostrate their specific involvement on chemoresistance. Recent data from our group indicated that OPNb and OPNc splicing isoforms activate PCa tumor progression features. Besides, PCa cells that overexpress these splice variants are more resistant to apoptosis induced by docetaxel (DXT). The current work aims to investigate the expression pattern of OPN splicing isoforms (OPN-SIs) and their relationships in DXT-PCa resistant cells. Total RNA has been extracted from PCa cell lines resistant to DXT (PC3-D8 and PC3-D12) and PC3 control cells with similar cell passages (PC3-Ag). Then, cDNa has been synthesized. OPN-SI expression levels were analyzed by quantitative real time PCR (qRT-PCR) using SYBR Green and isoform specific oligonucleotides. Relative expression levels were calculated using ΔΔCT method. Among the 3 OPN-SIs, we observed that OPNb and OPNc variants are overexpressed in relation to OPNa in DXT-PCa resistant cells PC3-D8 and PC3-D12, when compared to PC3-Ag. PC3-D12 cell line, resistant to higher DXT concentrations, expresses higher levels of OPNc, when compared to the other cell lines. Overall, our data further evidence that OPNb and OPNc overexpression in DXT-PCa resistant cells could be involved on resistance to DXT. Functional assays will be performed in order to investigate the molecular mechanisms by which these OPN-SIs mediate resistance to this chemotherapeutic drug. Citation Format: Mariana Concentino Menezes, Amanda O’Neil, Isabella Guimaraes, Letícia Rangel, Etel Rodrigues Pereira Gimba. Expression of osteopontin splicing isoforms in prostate cancer cells resistant to docetaxel. [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 2937.

In ovarian carcinoma (OC), invasive cell properties are supported by epithelial mesenchymal transition (EMT). We previously demonstrated that OPNc, one of the glycophosphoprotein osteopontin splicing isoforms (OPN-SI), activates OC progression. However , the involvement of these OPN-SI in EMT in OC cells has not previously been addressed. We aimed to evaluate the expression of OPN-SI in OC cells induced to EMT by TGFβ. ES2 and A2780 OC cell lines were treated with 10ng/ml of TGFβ in order to induce EMT. The expression of OPN-SI and EMT epithelial (E) (Claudin-3, Claudin-4 and E-cadherin) and mesenchymal (M) (slug, vimentin and N-cadherin) markers was tested by quantitative real time PCR. In both cell lines, OPNa is overexpressed in relation to OPNb and OPNc. Upon treatment with TGFβ, in A2780 cells we observed an upregulation of OPNc. Conversely, OPNa and OPNb were downregulated. In ES2 cells, TGFβ also induced OPNc upregulation, but OPNb expression is activated as well, while OPNa expression is inhibited. In order to validate EMT induction by TGFβ, we tested the expression of E and M markers. In A2780 cells, TGFβ induce the downregulation of E-cadherin, but Claudin-3 and Claudin-4 are upregulated. In ES2 cells, Claudin-3 expression is downregulated in this condition. After TGFβ treatment, in A2780 cells all M markers are downregulated, while in ES2 cells, only N-cadherin expression has been inhibited. Our data indicate that the expression of OPN-SI is modulated by EMT induced by TGFβ and that mostly the oncogenic OPNc is upregulated in this condition. Our data evidence that the expression of OPN-SI is modulated by a partial EMT phenotype induced by TGFβ, which is cell-type specific, and that OPN-SI, specially OPNc, could contribute to this key step on tumor progression in OC cells. Citation Format: Ingridy Celestino, Isabella S. Guimarães, Leticia B. A. Rangel, Etel R. P. Gimba. Osteopontin splicing isoforms expression is modulated by partial epithelial mesenchymal transition in ovarian carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A28.

Nucleus accumbens-associated protein 1 (NAC1) is a transcriptional co-regulator that lacks the DNA binding domain, thus relying on other co-factors to form higher-order transcription complexes. The present study aimed to elucidate the mechanism by which NAC1 modulates gene expression in OVCA. Nacc1-/- mice were euthanized, and spleens were collected. Promoter sequences were retrieved using Biostrings and BSgenome packages of Bioconductor 2.12. The position frequency matrix of BCL6 motif was obtained from Cistrome. BCL6 binding motifs on targeted promoters were identified by matchPWM, and log-odd scores calculated by PWMscoreStartingAt. For chromatin immunoprecipitation (ChIP), cells were fixed with 1% (w/v) paraformaldehyde prior to cross-linking with DTBP. Anti-BCL6, anti-NAC1 or rabbit IgG isotype were used for IP, and the genes of interest were amplified by qPCR. HEK293T cells were used for co-immunoprecipitaion (co-IP), and proteins were analyzed by Western blot. Luciferase reporter assays were performed in HeLa cells transfected with siNAC1 and/or siBCL6. Immunohistochemistry was done on tissue microarray (TMA) constituted of 51 primary ovarian high-grade serous carcinoma samples (HGSOC), using anti-NAC1 or anti-BCL6 antibodies. For microarray experiments, OVCAR5 cells were transfected with siNAC1 or siBCL6. Two-tailed t-test was used analyze data (means ± SD; p Citation Format: Alice Laschuk Herlinger, Min Gao, Ren-Chin Wu, Tian-Li Wang, Leticia B A Rangel, Ie-Ming Shih. NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in ovarian cancer (OVCA). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A80.

In the present study, we aimed to evaluate estrogen receptor ER-alpha status in 61 breast cancer cases using Sp1 and 1D5 monoclonal antibodies. Tissue array platforms were generated containing samples of breast cancer and positive controls that were assayed by immunohistochemistry applying monoclonal primary antibodies anti-ER alpha, SP1 and 1D5. We noted a high concordance rate (96.7%) between the referred antibodies. Moreover, we calculated the Kappa factor (0.921), indicating that 1D5 and SP1 provided overlapping ERα expression results. Indeed, we observed controversial results only in 2 samples studied, which were ER-negative when stained with 1D5 and ER-positive when assessed with SP1. Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively). However, concordance between the antibodies seems to be more accurate in higher PS values. An excellent IS correlation between antibodies was observed throughout the population (Spearman's CF, ρ=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged, with 1D5 always pointing to weaker staining than SP1. When calculating Spearman's CF of Total Score (TS) within the population, an excellent correlation between both the antibodies (ρ=0.9238) was noted. Nonetheless, the results were less concordant among the BC-positive cases (ρ=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p

In the last decades, the Oncology field has faced an era where the pace of biotechnological advances has promised improvements in cancer diagnosis and treatment in a truly impressive manner. In this context, the enlightenment of cancer biology and carcinogenesis mechanisms have enabled not only more accurate diagnosis of the disease, therefore guiding more specific and efficient therapeutic approaches, but also allowing the discovery of novel biomarkers to fight cancer with molecular targets. Regardless the referred progress in medicine, there is still low tumor responsiveness index to classic chemotherapy regimens, and an epidemiology scenario that shows an increase in cancer-related mortality rate over the years. According to American Cancer Society, in January of 2006, about 11.6 million living Americans had already developed cancer during lifetime. For the year of 2010, there are expected over 1.5 million new cancer cases diagnosis and about 1,500 cancer-related deaths daily in the USA. In the present chapter, the focus will be given to two major types of cancer affecting women’s health care: breast cancer(BC) and ovarian cancer(OVCA). Whereas BC accounts for near 23% of all cancers diagnosed and 13.7% of cancer-related deaths in women, OVCA, although not very incident (approximately 3.7% of cancer cases among women), correlates to an extremely high mortality rate of affected women (approximately 4.2% of cancer related deaths among women).

The aim of this work was to study the estrogen receptor alpha (ERS1) PvuII and XbaI gene polymorphisms prevalence in randomly selected women population from Rio de Janeiro and Espírito Santo states in Brazil by polymerase chain reaction restriction fragment lengths polymorphism (PCR_RFLP) methodology. It was shown that Rio de Janeiro women exhibited a significantly different prevalence of XbaI polymorphism comparing to Espirito Santo women. Nonetheless, similar prevalence of PvuII polymorphism was found in both women´s populations. Moreover, a strong linkage disequilibrium was observed between these SNPs reinforcing the hypothesis of differential pattern of inheritance observed on such populations.

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

Advances in our understanding of CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(Regs)) enabled the characterization of their activities in maintaining peripheral tolerance, preventing autoimmune diseases, and limiting chronic inflammatory diseases. Ironically, an effective action of these cells during tumor development can limit beneficial responses by suppressing immunity and limiting antitumor resistance, whereas one of the main functions of the immune system is to eliminate malignant cells. During the last years, the immunological role, mechanism of action, and clinical importance of these cells were profoundly characterized and the relationship between this subset of lymphocytes and cancerous cells arises as a key factor that influences tumor development. Recent insights obtained from clinical studies and experimental mouse models expand our perception of the potential role of T(Regs) in cancer treatment. In this review we describe the basic mechanisms of T(Reg) origin and differentiation, their potential role in cancer, as well as the future perspectives concerning the modulation of these cells as a potential approach for anticancer strategies.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, mostly due to the diagnosis of the disease at advanced stages and to chemoresistance to platinum- and taxane-based standard therapy. EOC comprises a heterogeneous group of diseases classified based on morphologic and molecular-genetic features. High grade serous ovarian cancer is the most prevalent EOC subtype, being followed by the clear cell subtype. Both EOC subtypes are vastly aggressive and frequently acquire the chemoresistant phenotype, thus challenging researchers and clinicians. Notably, hyperactivation of PI3K and MAPK pathways are frequently related to resistance to standard therapies in EOC, thus, metformin (MET) and everolimus (EVE) have emerged as novel therapeutic options against EOC. The present work aimed to investigate the action of MET and EVE, in vitro, in ES-2 (clear cell) and A2780 (serous) cell lines. Methods: Metabolic cell viability (MCV) assays were conducted after the treatment of the cells with MET or EVE in monotherapy and in association with paclitaxel (PAC) by the MTT method. Cell cycle progression was evaluated by flow cytometry/propidium iodide. Apoptosis was analyzed by the annexin V-FITC assay. Western blot was performed to investigate MEK/ERK and PI3K/AKT pathways. Findings: MCV of EOC lineages was reduced by MET and EVE in different concentrations. The combination of MET, at low and safe concentration (10uM), with PAC 100nM reduced the MCV in 56.55% for ES-2, and 71.38% for A2780. Similarly, EVE 0.06 nM and PAC 100 nM decreased the VCM in 66.4% and 73.38% for ES-2 and A2780, respectively. MET 10μm and EVE 0.06nM associated with PAC 12.5nM kept the MCV at levels comparable to PAC 100nM. These findings are of extreme clinical relevance as they reveal that the synergism between MET and EVE with PAC likely maintains the drug citotoxicity at lower concentrations than those associated to its severe and limiting side effects, as neurotoxicity. Annexin-FITC assay was conducted in ES-2 lineage treated with PAC alone and combined with MET or EVE. Low percentage of apoptotic/necrotic cells was observed, which suggests the occurrence of additional processes, such as autophagic cell death. EVE induced ES-2 cell cycle arrest; however, MET, at low concentration, did not cause the same effect. There were no significant cell cycle changes due to the combined treatment of ES-2 with PAC and MET or EVE. Western blot analysis demonstrated that the combination of EVE and PAC increased, whereas MET alone or in combination with PAC decreased the phosphorylation of ERK1/2 and AKT in ES2 cells. Conclusions: Our data suggest that MET or EVE exerts synergistic effect with PAC against EOC, thus opening an avenue to explore novel strategies against this highly lethal disease. Ongoing experiments are sought to elucidate the cellular pathways modulated by MET and EVE alone or in combination with PAC in EOC lineages, as well as the elicited cellular events and mechanisms. Citation Format: Nayara G. Tessarollo, Isabella S. Guimaraes, Taciane Ladislau, Ian Victor Silva, Leticia B. A. Rangel. Metformin and everolimus act synergistically with paclitaxel against ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2015-2548

Estrogen receptor α (ERα) is the defining and driving transcription factor in luminal breast cancers and genes modulated by ERα can dictate cell growth and endocrine response. Nonetheless, genomic mechanisms which govern ERα-driven tumorigenesis, acquisition of chemoresistant phenotype and tumor metastases remain elusive. Recently, scientists have explored to shed light on ER regulated genomic events in primary breast cancer with divergent clinical outcome and in distant ERα+ metastases. Mapping of genome-wide ER binding events by chromatin immunoprecipitation followed by high-throughput sequencing revealed differential FoxA1-mediated ERα-chromatin binding programming that results in predictive gene signatures exclusive for ERα+ breast cancer clinical outcome, and is characterized by remarkable intensification of ERα binding signal in tumors that progress towards a poor prognosis [1,2]. Furthermore, ERα-chromatin interactions occur regardless of tumor endocrine therapy sensitivity. Nevertheless, there is differentially stronger ERα binding signal in tamoxifen resistant in comparison with tamoxifen sensitive lineages [1]. Although the mechanisms underlying ERα binding plasticity in breast cancer remain to be elucidated, the influence of specific stimuli, as those triggered by growth factors pathways [3,4], may result in differential ERα binding patterns that regulate gene expression programs, sensitivity to endocrine therapy and overall clinical outcome in ERα+ breast tumors. It is worthwhile to emphasize that the only DNA motif found enriched in the core of ER binding events is the estrogen response element (ERE) [1]. Of note, innovative model in ERα-driven tumorigenesis and cancer aggressiveness has emerged from the concept of the tag-team model of gene expression in luminal breast cancer, according to which amplified distant EREs (DEREs) coordinately and remotely modulates the transcription of distant genes through long-rage chromatin interactions [5]. The model hypothesis highlights DERE axes as hot spots for concomitant and aberrant genome amplification in luminal breast cancer; thus coordinately and persistently deregulating target transcriptome, including co-amplification of oncogenes and repression of tumor-suppressor loci for cancer development and endocrine therapy resistance. Using integrated next-generation sequencing approaches, two densely ERα-bound DERE regions, frequently amplified in ERα+ luminal breast cancers, were mapped on chromosomes 17q23 and 20q13 [5]; genomic amplification of which has been associated with endocrine therapy relapse and overall poor prognosis in breast cancer [6]. Moreover, integration of 3C dataset with published time-course study of gene expression revealed 95 loci remotely interacting with 20q13 DEREs, 38 genes with 17q23 DEREs, and 46 estrogen-responsive targets [5]. Interestingly, a strong body of evidences has demonstrated the novel role of the DEREs targeted genes THRAP1 and ZIM2 as tumor suppressors in luminal breast cancer. It is reasonable to speculate that the increased frequency of chromatin interactions might play a role to elicit epigenetic repression of DERE-regulated genes. The aforementioned phenomenon derives from genomic alterations induced by chronic estrogenic exposure of mammary cells that persist in breast progenitor cells [5], and progressively accumulate in malignant differentiating cells, in agreement with previous findings that points to the intensification of the ERα binding signal and ERE amplification during breast cancer worsening prognosis [1,2]. Precisely, these findings open new avenues to explore amplified DEREs in 17q23 and 20q13 as potential prognostic markers in luminal breast cancer, based on the rationale that the aberrant amplification of DEREs may enable functionality of residual estrogen/ERα regardless of the administration of selective ER blockers, such as tamoxifen [5]. This concept, at least partially, can explain the benefit of using aromatase inhibitors in post-menopausal women, which present the higher incidence of luminal breast cancer. The inhibition of the main source of estrogen production in these patients may prevent the E2/ERα-induced anomalous amplification of DEREs. On the other hand, the amplified DEREs axes and the tag-team model of gene expression modulation in luminal breast cancer currently challenges the investigators to determine the precise factors and mechanisms that trigger the malignant transformation of normal progenitor mammary cells. Amplified DNA regulatory elements may result from sustained amplification of DERE-DERE interactions that may intensify chromatin interaction within regions harboring clustered breakpoints, exquisitely prone to genomic rearrangements, as a result of genomic instability [5,7]. During neoplastic transformation, defects in double-strand break repair may destabilize these physical interactions, promoting insertions and self-duplication of 20q13 DERE clusters, for example, into regions of seven derivative chromosomes [5]. In contrast to this concept, it is puzzling that mammary cells sustain the benign phenotype in the majority of women regardless of the estrogenic milieu. At present, it is also important to evaluate the occurrence of aberrant DERE amplification and its peculiarities in controlling target genes expression in ovarian cancer, commonly lethal and tamoxifen resistant. Nonetheless, cumulative evidence hint the potential roles of the amplified distant DNA response elements axes and the tag-team model of gene expression modulation in hormone-driven carcinogenesis and cancer progression. More importantly, knowledge gained out of the study [5] should be instrumental for further understanding of cancer genomics and bringing of innovative interventions to control cancer.

Tumor formation in immunocompetent hosts is believed to be dependent on the ability of tumor cells to evade the immune system, as suggested by the alterations of expression of the major histocompatibility complex (MHC) and related molecules in a number of cancers. Our previous serial analysis of gene expression (SAGE) study revealed that HLA-DRA (encoding the alpha chain of HLA-DR) is one of the most highly overexpressed genes in ovarian cancer. This finding was unanticipated, as overexpression of MHC molecules would be expected to increase tumor immunogenicity, therefore compromising tumor growth. We have now examined the expression of HLA-DR alpha chain in ovarian and a variety of other cancers using tissue arrays and found it overexpressed in a majority of the cancer tissues investigated. In contrast, the HLA-DR beta chain, which together with the alpha chain forms the functional HLA-DR complex, was not frequently found expressed in cancer, resulting to a lack of mature HLA-DR in these tissues. Interestingly, HLADRA and HLADRB transcripts were both found expressed in many other cancer types, including ovarian cancer, suggesting that the downregulation of HLADR beta chain is a post-transcriptional or post-translational mechanism. In addition, we observed high levels of the invariant chain (Ii/CD74) expression in both the cytoplasm and plasma membrane of ovarian tumor cells, possibly contributing to the lack of mature HLA-DR protein expression. Interestingly, we found that IFN-gamma could induce mature HLA-DR at the surface of normal ovarian cells, while this ability was reduced in tumor cells. Together, these data suggest that, while ovarian tumors overexpress HLA-DR alpha, perhaps as a result of inflammatory events in the tumor microenvironment, the tumor cells may have compensatory mechanisms to reduce the production of functional MHC class II molecules, thus reducing immunogenicity and favoring tumor growth. In addition, because of its ubiquitous expression in ovarian and other cancers, HLA-DR alpha may represent a novel biomarker for malignancy.

The mechanisms of drug resistance in cancer are poorly understood. Serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive cells revealed many differentially expressed genes. Remarkably, many ECM genes were elevated in cisplatin-resistant cells. COL6A3 was one of the most highly upregulated genes, and cultivation of cisplatin-sensitive cells in the presence of collagen VI protein promoted resistance in vitro. Staining of ovarian tumors with collagen VI antibodies confirmed collagen VI expression in vivo and suggested reorganization of the extracellular matrix in the vicinity of the tumor. Furthermore, the presence of collagen VI correlated with tumor grade, an ovarian cancer prognostic factor. These results suggest that tumor cells may directly remodel their microenvironment to increase their survival in the presence of chemotherapeutic drugs.

Background: Lung cancer (LC) represents a major health challenge worldwide, being the leading cause of cancer-related deaths in both men and women. SLC34A2 is a member of the solute carrier family and encodes the type IIb sodium phosphate cotransporter (NaPi-IIb). SLC34A2 expression is reduced by ten-fold in LC samples compared to normal lung. Estrogen is a known risk factor for LC in women, and a relationship between its action and SLC34A2/NaPi-IIb super-expression in certain cell lines has been proposed. Furthermore, based on the central role of PKC isoenzymes in carcinogenesis, and on studies showing that administration of cAMP stimulates the NaPi-IIb mRNA expression in adult rats alveolar type II cells, this work aimed to evaluate the correlation between PKA (cAMP mediator), PKC, estrogen signaling pathways and SLC34A2/NaPi-IIb expression levels in human LC cells. Methods: For both LC cell lines studied, A549 and H460, the effects of PKC and PKA activating and inhibitory reagents (PMA, calphostin C, dB cAMP and KT 5720) were tested in relation to SLC34A2/NaPi-IIb expression. Additionally, a dose-response curve of 17-beta-estradiol was performed for the expression of SLC34A2/NaPi-IIb in A549 cell line. SLC34A2/NaPi-IIb gene expression was analyzed by qRT-PCR, using SYBR Green PCR Master Mix as the detection system. Relative quantification of gene expression was performed by calculating the delta-delta Ct using two housekeeping genes: GAPDH and Beta-Actin. Findings: In our analysis we found that PKC and PKA did not influence the expression of SLC34A2/NaPi-IIb in LC cell line evaluated. There was, however, a considerable increase in the expression of this gene when treated with calphostin C, probably by a mechanism independent of its classic role in the PKC inhibition. There was also a trend of decreased SLC34A2/NaPi-IIb gene expression in LC cell line following the treatment with 17 beta-estradiol, and SLC34A2/NaPi-IIb - which expression is already reduced in LC - undergoes a greater reduction in its relative expression. Interpretation: We believe that maintaining the reduced expression of SLC34A2/NaPi-IIb should provide benefits to LC cells. Calphostin C induces apoptosis in several tumor cell lines by mechanisms not yet fully elucidated. Thus, we suggest a possible involvement of SLC34A2/NaPi-IIb in this pro-apoptotic pathway, which corroborates the fact that LC tumors present a reduced SLC34A2/NaPi-IIb expression compared to normal lung. Likewise, based on our findings, we believe that the inclusion of selective Estrogen receptor modulators and aromatase inhibitors in the routine clinical practice of LC might help to control the disease that is still the leading cause of cancer-related deaths worldwide. Citation Format: Murilo F. Cerri, Lucas C d Rezende, Marcela F. Paes, Klesia P. Madeira, Renata D. Daltoe, Nayara G. Tessarollo, Ian V. Silva, Leticia B a Rangel. Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators. [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 463. doi:10.1158/1538-7445.AM2014-463