The impact of lipid metabolism on breast cancer: a review about its role in tumorigenesis and immune escape.

BACKGROUND: Breast cancer (BC) is the second most frequent type of cancer in the world and most common among women, configuring a major challenge to global health. BC is a complex and heterogeneous disease that can be subdivided into distinct tumor types based on the expression of molecular markers predicting patient outcomes and response to therapy. A growing number of studies have tried to expand the known markers by investigating the association of altered lipid metabolism with BC immune escape, progression, and metastasis. In this review, we describe the metabolic peculiarities of each BC subtype, understanding how this influences its aggressiveness and identifying whether these intrinsic vulnerabilities of each subtype can play a role in therapeutic management and may affect immune system cells in the tumor microenvironment. CONCLUSION: The evidence suggests so far that when changes occur in lipid pathways, it can affect the availability of structural lipids for membrane synthesis, lipid synthesis, and degradation that contribute to energy homeostasis and cell signaling functions. These findings will guide the next steps on the path to understanding the mechanisms underlying how lipids alterations are related to disparities in chemotherapeutic response and immune escape in BC. Video Abstract.

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance.

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.

Role of APOE Gene in Bone Mineral Density and Incidence of Bone Fractures in Brazilian Postmenopausal Women.

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.

Pharmacogenomic diversity of tamoxifen metabolites and estrogen receptor genes in Hispanics and non-Hispanic whites with breast cancer.

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.

Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells.

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.

Atorvastatin improves cisplatin sensitivity through modulation of cholesteryl ester homeostasis in breast cancer cells.

BACKGROUND: Acquired treatment resistance is a significant problem in breast cancer management, and alterations in lipid metabolism have been proposed to contribute to the development of drug resistance as well as other aspects of tumor progression. The present study aimed to identify the role of cholesterol metabolism in MCF-7 and MDA-MB-231 breast cancer cell response to cisplatin (CDDP) treatment in the acute setting and in a model of CDDP resistance. METHODS: MCF-7 (luminal A), MDA-MB-231 (triple-negative) and CDDP-resistant MDA-MB-231 (MDACR) cell lines were grown in the presence or absence of CDDP in combination with atorvastatin (ATV), lipid depletion or low-density lipoprotein loading and were analyzed by a variety of biochemical and radiometric techniques. RESULTS: Co-administration of CDDP and ATV strongly reduced cell proliferation and viability to a greater extent than CDDP alone, especially in MDA-MB-231 cells. These findings were associated with reduced cholesteryl ester synthesis and storage in MDA-MB-231 cells. In MDACR cells, acetyl-CoA acetyltransferase 1 (ACAT-1) was upregulated compared to naïve MDA-MB-231 cells and ATV treatment restored CDDP sensitivity, suggesting that aberrant ACAT-1 expression and associated changes in cholesterol metabolism contribute to CDDP resistance in MDA-MB-231 cells. CONCLUSION: These findings indicate that the elevated susceptibility of MDA-MB-231 cells to co-administration of CDDP and ATV, is associated with an increased reliance on cholesteryl ester availability. Our data from these cell culture-based studies identifies altered cholesterol homeostasis as an adaptive response to CDDP treatment that contributes to aggressiveness and chemotherapy resistance.

Two distinct mechanisms underlie estrogen-receptor-negative breast cancer susceptibility at the 2p23.2 locus.

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.

Fat-storing multilocular cells expressing CCR5 increase in the thymus with advancing age: potential role for CCR5 ligands on the differentiation and migration of preadipocytes.

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.

Characterization of novel human ovarian cancer-specific transcripts (HOSTs) identified by serial analysis of gene expression.

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.

Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas.

PURPOSE: Claudin proteins represent a large family of integral membrane proteins crucial for tight junction (TJ) formation and function. Claudins have been shown to be up-regulated in various cancers and have been suggested as possible biomarkers and targets for cancer therapy. Because claudin-3 and claudin-4 have been proposed to be expressed in epithelial ovarian cancer, we have performed a detailed analysis of CLDN3 and CLDN4 expression in a panel of ovarian tumors of various subtypes and cell lines. We also investigated whether high expression of claudin-3 and claudin-4 was associated with TJ function in ovarian cancer cells. EXPERIMENTAL DESIGN: RNA was obtained from a panel of 39 microdissected epithelial ovarian tumors of various histological subtypes for real-time reverse transcription-PCR analysis. In addition, a total of 70 cases of ovarian carcinomas, ovarian cysts, and normal ovarian epithelium from a tissue array were analyzed by immunohistochemistry. Finally, a panel of cell lines was used for Western analysis of claudin expression and TJ permeability studies. RESULTS: Although expressed at low levels in some normal human tissues, including the ovary, CLDN3 and CLDN4 are highly up-regulated in epithelial ovarian cancers of all subtypes. Immunohistochemical analyses using our ovarian tissue array confirmed the high level of expression of claudin-3 and claudin-4 in the majority of ovarian carcinomas, including many tumors exhibiting cytoplasmic staining. Ovarian cystadenoma did not frequently overexpress these proteins, suggesting that the expression of these proteins is associated with malignancy. In ovarian cancer cell lines, claudin-3 and claudin-4 expression was not associated with functional TJs as measured by transepithelial electrical resistance. CONCLUSIONS: These results show that CLDN3 and CLDN4 are frequently up-regulated in ovarian tumors and cell lines and may represent novel markers for this disease. Overexpression of these genes in ovarian cancer also suggests interesting scenarios for the involvement of TJ in tumorigenesis. A better knowledge of the mechanisms underlying ovarian tumorigenesis will likely result in the development of novel approaches for the diagnosis and therapy of this deadly disease.

Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.

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 17ß-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.

Anomalous expression of the HLA-DR alpha and beta chains in ovarian and other cancers.

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.

Prescription of psychoactive drugs in patients attended by the SUS at Manhuaçu - MG (Brazil).

OBJECTIVE: 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 Manhuaçu, 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. CONCLUSIONS: 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.

Prescription of psychoactive drugs in patients attended by the SUS at Manhuaçu - MG (Brazil) / Prescripción de medicamentos psicoactivos en pacientes atendidos por el SUS en Manhuaçu - MG (Brasil)

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 Manhuaçu, 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 (AU)

Objetivos: En este estudio presentamos el desarrollo de una base de datos de medicamentos psicoactivos dispensados a pacientes atendidos por el Sistema Público de Salud brasileño (SUS) en la ciudad de Manhuaçu, Minas Gerais y el patrón de prescripción en esta ciudad. Métodos: Se investigó a 827 pacientes bajo tratamiento con psicoactivos y atendidos por el SUS y se recogió información como género, nivel de educación, edad, estado civil. Los datos recogidos se analizaron para dibujar el perfil de los pacientes y la dispensación y la información se utilizó para crear el patrón de uso de psicoactivos en la ciudad. Resultados: Las mujeres sumaron el 67,2% de la población y la edad parecía influenciar positivamente el uso de medicamentos psicoactivos. Las benzodiazepinas y los antidepresivos estaban entre los medicamentos más prescritos, especialmente después de los 20 años, mientras que en la población más joven los antipsicóticos y los antiepilépticos fueron los medicamentos más prescritos. Los antiepilépticos y los estabilizadores del ánimo parecían ser prescritos principalmente a hombres y mujeres solteros. Conclusión: Los datos personales como género, edad y estado civil están relacionados con la dispensación de medicamentos psicoactivos. Los datos recogidos servirán como apoyo para la actuación de los farmacéuticos responsables de la dispensación de psicoactivos en el municipio (AU)