Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.

For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.

Exposure to anticancer drugs modulates the expression of ACSL4 and ABCG2 proteins in adrenocortical carcinoma cells.

Adrenocortical carcinoma (ACC) is a rare and malignant disease, with more than 50 % of patients developing hormone-secreting tumors. These tumors are genetically heterogeneous and potentially lethal, as metastasis is often underway at the time of diagnosis. While chemoresistance can be multifactorial, Acyl CoA synthetase 4 (ACSL4) is known to contribute to the generation of highly aggressive cellular phenotypes, while increased expression and activity of multidrug transporters such as ATP-binding cassette subfamily G member 2 (ABCG2) are known to play a key role. Therefore, the objective of this work was to determine changes in the expression of ACSL4 and ABCG2 in ACC cell lines after exposure to antitumor drugs. Bioinformatics analysis of public database GSE140818 revealed higher ACSL4 and ABCG2 expression in HAC15 cells resistant to mitotane when compared to wild type cells. In addition, our studies revealed an increase in ACSL4 and ABCG2 expression in lowly aggressive H295R cells undergoing early treatment with non-lethal concentrations of mitotane, doxorubicin and cisplatin. Comparable results were obtained in lowly aggressive breast cancer cells MCF-7. The increase in ACSL4 and ABCG2 expression favored tumor cell viability, proliferation and compound efflux, an effect partially offset by ACSL4 and ABCG2 inhibitors. These results provide relevant data on the undesired molecular effects of antitumor drugs and may fuel future studies on patients' early response to antitumor treatment.

Acyl-CoA synthetase-4 is implicated in drug resistance in breast cancer cell lines involving the regulation of energy-dependent transporter expression.

Acyl-CoA synthetase-4 (ACSL4) is an enzyme implicated in estrogen receptor α (ERα) negative regulation and hormone therapy resistance in breast cancer. In addition, ACSL4 has been associated to certain types of hormone resistance in prostate cancer. Chemotherapeutic treatment of disseminated breast cancer is usually faced with therapy resistance associated to ATP-binding cassette (ABC) transporter expression, which detect and eject anti-cancer drugs from cells. In this context, the aim of the present work was to study the role of ACSL4 in anti-cancer drug resistance and the involvement of ABC transporters in the underlying mechanisms. To this end, we used MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, which overexpress ACSL4, and control line MCF-7 Tet-Off empty vector, MDA-MB-231 shRNA ACSL4 and MDA-MB-231 wild type cells. Assays were conducted on cell viability (MTT), cell proliferation (BrdU), drug efflux (flow cytometry), ACSL4-responsive drug resistance ABC transporter genes (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot). Higher survival rates upon chemotherapeutic treatment were obtained in MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, an effect counteracted by doxycycline- or shRNA-induced ACSL4 inhibition, respectively. A synergic effect of ACSL4 inhibitor triacsin C and chemotherapeutic drugs was observed on the inhibition of MDA-MB-231 wild type cell proliferation. MCF-7 Tet-Off/ACSL4 cells showed greater doxorubicin, Hoechst 33342 and calcein AM efflux. In contrast, MDA-MB-231 shRNA ACSL4 cells evidenced inhibition of chemotherapeutic drug efflux. ABCG2, ABCC4, and ABCC8 were identified as ACSL4-responsive drug resistance genes whose expression was increased in MCF-7 Tet-Off/ACSL4 cells but inhibited in MDA-MB-231 shRNA ACSL4 cells. Further cell survival assays in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment showed greater participation of ABCG2 in anti-cancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapamycin-induced mTOR inhibition synergically inhibited proliferation and reduced ABCG2 expression in cells overexpressing ACSL4. In sum, ACSL4 may be regarded as a novel therapeutic target regulating the expression of transporters involved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival.

Spectrum of BRCA1/2 variants in 940 patients from Argentina including novel, deleterious and recurrent germline mutations: impact on healthcare and clinical practice.

BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements. Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described. IN CONCLUSION: a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina.

BRCA1 And BRCA2 analysis of Argentinean breast/ovarian cancer patients selected for age and family history highlights a role for novel mutations of putative south-American origin.

BACKGROUND: The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been scarcely investigated outside Europe and North America, with few reports for South America, where Amerindian founder effects and recent multiracial immigration are predicted to result in high genetic diversity. We describe here the results of BRCA1/BRCA2 germline analysis in an Argentinean series of breast/ovarian cancer patients selected for young age at diagnosis or breast/ovarian cancer family history. METHODS: The study series (134 patients) included 37 cases diagnosed within 40 years of age and no family history (any ethnicity, fully-sequenced), and 97 cases with at least 2 affected relatives (any age), of which 57 were non-Ashkenazi (fully-sequenced) and 40 Ashkenazi (tested only for the founder mutations c.66_67delAG and c.5263insC in BRCA1 and c.5946delT in BRCA2). DISCUSSION: We found 24 deleterious mutations (BRCA1:16; BRCA2: 8) in 38/134 (28.3%) patients, of which 6/37 (16.2%) within the young age group, 15/57 (26.3%) within the non-Ahkenazi positive for family history; and 17/40 (42.5%) within the Ashkenazi. Seven pathogenetic mutations were novel, five in BRCA1: c.1502_1505delAATT, c.2626_2627delAA c.2686delA, c.2728 C > T, c.3758_3759delCT, two in BRCA2: c.7105insA, c.793 + 1delG. We also detected 72 variants of which 54 previously reported and 17 novel, 33 detected in an individual patient. Four missense variants of unknown clinical significance, identified in 5 patients, are predicted to affect protein function. While global and European variants contributed near 45% of the detected BRCA1/2 variation, the significant fraction of new variants (25/96, 26%) suggests the presence of a South American genetic component. This study, the first conducted in Argentinean patients, highlights a significant impact of novel BRCA1/2 mutations and genetic variants, which may be regarded as putatively South American, and confirms the important role of founder BRCA1 and BRCA2 mutations in Argentinean Ashkenazi Jews.

High-risk human papilloma virus infection, tumor pathophenotypes, and BRCA1/2 and TP53 status in juvenile breast cancer.

Juvenile breast cancer is rare and poorly known. We studied a series of five breast cancer patients diagnosed within 25 years of age that included two adolescents, 12- and 15-years-old, and 3 young women, 21-, 21-, and 25-years-old, respectively. All cases were scanned for germline mutations along the entire BRCA1/2 coding sequences and TP53 exons 4-10, using protein truncation test, denaturing high performance liquid chromatography and direct sequencing. Paraffin-embedded primary tumors (available for 4/5 cases), and a distant metastasis (from the 15-years-old) were characterized for histological and molecular tumor subtype, human papilloma virus (HPV) types 16/18 E6 sequences and tumor-associated mutations in TP53 exons 5-8. A BRCA2 germline mutation (p.Ile2490Thr), previously reported in breast cancer and, as compound heterozygote, in Fanconi anemia, was identified in the 21-year-old patient diagnosed after pregnancy, negative for cancer family history. The tumor was not available for study. Only germline polymorphisms in BRCA1/2 and/or TP53 were detected in the other cases. The tumors of the 15- and 12-years-old were, respectively, classified as glycogen-rich carcinoma with triple negative subtype and as secretory carcinoma with basal subtype. The tumors of the 25-year-old and of the other 21-year-old were, respectively, diagnosed as infiltrating ductal carcinoma with luminal A subtype and as lobular carcinoma with luminal B subtype. No somatic TP53 mutations were found, but tumor-associated HPV 16 E6 sequences were retrieved from the 12- and 25-year-old, while both HPV 16 and HPV 18 E6 sequences were found in the tumor of the 15-year-old and in its associated metastasis. Blood from the 15- and 25-year-old, diagnosed with high-stage disease, resulted positive for HPV 16 E6. All the HPV-positive cases were homozygous for arginine at TP53 codon 72, a genotype associated with HPV-related cancer risk, and the tumors showed p16(INK4A) immunostaining, a marker of HPV-associated cancers. Notably menarche at 11 years was reported for the two adolescents, while the 25-year-old was diagnosed after pregnancy and breast-feeding. Our data suggest that high-risk HPV infection is involved in a subset of histopathologically heterogeneous juvenile breast carcinomas associated with menarche or pregnancy and breast-feeding. Furthermore we implicate BRCA2 in a juvenile breast carcinoma diagnosed at 21 years of age, 4 years after an early full-term pregnancy, in absence of cancer family history.