Ginés Luengo-Gil

Association of anthracycline-related cardiac histological lesions with NADPH oxidase functional polymorphisms.

OBJECTIVE Treatment with anthracyclines may cause cardiac dysfunction, but the sequence of anthracycline-induced heart lesions has been incompletely characterized. NADPH oxidase, a key mediator of oxidative cardiac damage and remodeling, modulates anthracycline clinical cardiotoxicity. Our aim was to determine which cardiac histological lesions are specifically induced by anthracycline treatment and to investigate the role of NADPH functional genetic polymorphisms in their development. PATIENTS AND METHODS Using a retrospective case-control design, we evaluated cardiac histological lesions and NADPH genotype (polymorphisms rs1883112, rs4673, and rs13058338) in 97 consecutive decedents with a cancer diagnosis (48 treated with anthracyclines). RESULTS Myocytolysis (60%), patched myocardial necrosis (19%), and myocardial fibrosis (diffuse and patched; 62% and 23%, respectively) were associated with anthracycline treatment. In patients receiving anthracyclines, NADPH oxidase polymorphism rs4673 protected against focal myocardial necrosis (odds ratio [OR], 0.11; 95% confidence interval [CI], 0.20-0.63) whereas rs1883112 was strongly associated with cardiac fibrosis (OR, 5.11; 95% CI, 1.59-16.43), which was present in all homozygotes. CONCLUSION Anthracyclines induce a cardiac remodeling pattern characterized by interstitial or patched fibrosis. The contribution of the functionally relevant NADPH polymorphisms rs1883112 and rs4673 to anthracycline-related heart lesions provides a plausible explanation for their modulation of cardiotoxicity. If confirmed, these findings may lead to better individualized strategies for early detection and prevention of anthracycline cardiotoxicity.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Significance Vesicle fusion is an important event in neuronal transmission and endocrine cell secretion. A myriad of proteins containing double C2 domains are involved in this complex process; however, how Ca2+ and the different types of membrane lipids regulate their function is still not well understood. In this work, we provide structural insights to explain the ability of different C2 domains to interact with Ca2+ and PI(4,5)P2 and demonstrate the existence of a specific PI(4,5)P2-binding motif that provides these domains with specific properties to interact with the membrane and initiate vesicle fusion. We also demonstrate a unique molecular mechanism conferring their specificity for the different phosphoinositides, which resides in additional amino acidic residues surrounding the key interacting lysines. Proteins containing C2 domains are the sensors for Ca2+ and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca2+-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver

High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Leukemic transformation driven by an ASXL1 mutation after a JAK2V617F-positive primary myelofibrosis: clonal evolution and hierarchy revealed by next-generation sequencing

We have characterized the molecular changes underlying the transformation of a JAK2V617F+-myelofibrosis with trisomy 8, into a JAK2V617F-negative leukemia. Leukemic clone did not carry JAK2V617F mutation, but showed ASXL1 mutation (R693X). This mutation was identified in a low percentage at diagnosis by next-generation sequencing. Using this technology in serial specimens during the follow-up, we observed a progressive expansion of the ASXL1-mutated minor clone, whereas the JAK2V617F+-clone carrying trisomy 8 decreased. Hematologic progression occurred simultaneously with an ASXL1-R693X-negative lung-cancer. This is the first report showing a clear association between the expansion of an ASXL1-mutated clone and the leukemic transformation of myelofibrosis.

Role of Genetic Polymorphisms in NFKB-Mediated Inflammatory Pathways in Response to Primary Chemoradiation Therapy for Rectal Cancer

PURPOSE To investigate whether polymorphisms of genes related to inflammation are associated with pathologic response (primary endpoint) in patients with rectal cancer treated with primary chemoradiation therapy (PCRT). METHODS AND MATERIALS Genomic DNA of 159 patients with locally advanced rectal cancer treated with PCRT was genotyped for polymorphisms rs28362491 (NFKB1), rs1213266/rs5789 (PTGS1), rs5275 (PTGS2), and rs16944/rs1143627 (IL1B) using TaqMan single nucleotide polymorphism genotyping assays. The association between each genotype and pathologic response (poor response vs complete or partial response) was analyzed using logistic regression models. RESULTS The NFKB1 DEL/DEL genotype was associated with pathologic response (odds ratio [OR], 6.39; 95% confidence interval [CI], 0.78-52.65; P=.03) after PCRT. No statistically significant associations between other polymorphisms and response to PCRT were observed. Patients with the NFKB1 DEL/DEL genotype showed a trend for longer disease-free survival (log-rank test, P=.096) and overall survival (P=.049), which was not significant in a multivariate analysis that included pathologic response. Analysis for 6 polymorphisms showed that patients carrying the haplotype rs28362491-DEL/rs1143627-A/rs1213266-G/rs5789-C/rs5275-A/rs16944-G (13.7% of cases) had a higher response rate to PCRT (OR, 8.86; 95% CI, 1.21-64.98; P=.034) than the reference group (rs28362491-INS/rs1143627-A/rs1213266-G/rs5789-C/rs5275-A/rs16944-G). Clinically significant (grade ≥2) acute organ toxicity was also more frequent in patients with that same haplotype (OR, 4.12; 95% CI, 1.11-15.36; P=.037). CONCLUSIONS Our results suggest that genetic variation in NFKB-related inflammatory pathways might influence sensitivity to primary chemoradiation for rectal cancer. If confirmed, an inflammation-related radiogenetic profile might be used to select patients with rectal cancer for preoperative combined-modality treatment.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase.

Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an anti-inflammatory, anti-angiogenic, anti-viral and anti-apoptotic protein. Here, we report a novel function of antithrombin as a modulator of tumor cell migration and invasion. Antithrombin inhibited enteropeptidase on the membrane surface of HT-29, A549 and U-87 MG cells. The inhibitory process required the activation of antithrombin by heparin, and the reactive center loop and the heparin binding domain were essential. Surprisingly, antithrombin non-covalently inhibited enteropeptidase, revealing a novel mechanism of inhibition for this serpin. Moreover, as a consequence of this inhibition, antithrombin was cleaved, resulting in a molecule with anti-angiogenic properties that reduced vessel-like formation of endothelial cells. The addition of antithrombin and heparin to U-87 MG and A549 cells reduced motility in wound healing assays, inhibited the invasion in transwell assays and the degradation of a gelatin matrix mediated by invadopodia. These processes were controlled by enteropeptidase, as demonstrated by RNA interference experiments. Carcinoma cell xenografts in nude mice showed in vivo co-localization of enteropeptidase and antithrombin. Finally, treatment with heparin reduced experimental metastasis induced by HT29 cells in vivo. In conclusion, the inhibition of enteropeptidase by antithrombin may have a double anti-tumor effect through inhibiting a protease involved in metastasis and generating an anti-angiogenic molecule.

Angiogenic role of miR-20a in breast cancer

Background Angiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma. Methods Tube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples. Results In vitro angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (p = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (p<0.001). This association was independent of tumor subtype and VEGFA expression. Conclusions Transfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.

Transcriptomic rationale for synthetic lethality-targeting ERCC1 and CDKN1A in chronic myelomonocytic leukaemia

Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high‐throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA‐sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34+ bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down‐regulation to be specific to CMML compared with other related disorders. Chromatin‐regulator mutated cases showed decreased BAP1 dosage. We validated a significant over‐expression of the double strand break‐fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.

Regulation of TFPIα expression by miR-27a/b-3p in human endothelial cells under normal conditions and in response to androgens

The increased risk of cardiovascular events in older men is multifactorial, but the significant reduction of testosterone levels has been involved. As this hormone regulates the expression of TFPI by unknown mechanisms, we aimed to evaluate the role of miRNAs in the regulation of TFPIα expression under normal conditions and in response to androgens. In silico studies allowed the selection of 4 miRNAs as potential TFPIα regulators. Only miR-27a/b-3p significantly reduced TFPIα expression in two endothelial cell lines. Luciferase assays demonstrated a direct interaction between miR-27a/b-3p and TFPI 3′UTR. Ex vivo analysis of TFPI and miRNA levels in 74 HUVEC samples from healthy subjects, showed a significant and inverse correlation between TFPI and miR-27a-3p. Moreover, anticoagulant activity of TFPIα from cells supernatants decreased ~30% with miR-27a/b-3p and increased ~50% with anti-miR-27a/b-3p. Interestingly, treatment of EA.hy926 with a physiological dose of dihydrotestosterone (30 nM) significantly increased (~40%) TFPIα expression with a parallel decreased (~50%) of miR-27a/b-3p expression. In concordance, increased levels of miR-27a/b-3p normalized the up-regulation induced by testosterone. Our results suggest that testosterone is a hinge in miR-27/TFPIα regulation axis. Future studies are needed to investigate whether testosterone variations are involved in a miR-27/TFPIα dysregulation that could increase the cardiovascular risk.

Abstract P5-09-06: Decrease of tumor F3 expression after neoadjuvant chemotherapy associates to lower survival in breast cancer

Background: Pathologic complete response (pCR) is the main prognostic factor after neoadjuvant chemotherapy (nCT) for breast cancer (BC). However, in cases without pCR, additional prognostic biomarkers are needed for subsequent prognostic and therapeutic stratification of patients. Tissue factor (F3) is the protease initiator of blood coagulation cascade and is expressed in solid tumors including BC. F3 oncogenic functions derive both from coagulation activation and from its cytoplasmic domain, although they are not well known yet. Prognostic impact of F3 circulating levels and tumor expression seems to be variable, and no studies evaluating F3 expression changes in the residual tumor after nCT are available. Our aim was to evaluate chemotherapy-mediated changes in F3 mRNA levels and their prognostic value in BC treated with nCT. Methods: RNA was isolated from FFPE samples of pre- and post-CT tumors. Post-CT F3 levels were analyzed only in patients without primary tumor pCR. Quantification of F3 was performed by RT-qPCR. F3 expression was categorized as no expression or above/below median expression. Change in expression levels (ΔF3), defined as pre-nCT minus post-nCT, was categorized by value of change (positive vs. negative). Association of F3 levels with clinical and pathological characteristics and analysis of paired samples was evaluated with non-parametric tests. Kaplan-Meier curves, log-rank test and Cox proportional hazard regression multivariate models were used for survival analysis. To externally validate our results, we also studied the correlation between F3 and the rest of the set of genes in the TCGA database. The best 150 directly and 150 inversely correlated genes (r >0.3 and Results: We included 108 consecutive women with invasive BC, mostly with stages IIB or IIIA-C; Her2+: 25.0%, triple negative: 22.2%. After nCT including anthracyclines and taxanes, pCR rate was 19.4%. nCT significantly increased F3 expression (p Conclusion: Our data demonstrate that nCT consistently increases tumor F3 expression. However, those cases with decreased expression of F3 after chemotherapy show poor overall survival rates. Functional analysis in our series and in public databases demonstrate that decreased F3 mRNA expression correlates with an increased mitotic activity, suggesting that low F3 mRNA levels could be a marker of active and resistant to treatment tumors. Citation Format: Gines Luengo-Gil, Enrique Gonzalez-Billalabeitia, Rocio Gonzalez-Conejero, Asuncion Chaves-Benito, Elena Garcia-Martinez, Gloria Soler-Sanchez, Elisa Garcia-Garre, Lorena Velazquez, Vicente Vicente, Francisco Ayala de la Pena. Decrease of tumor F3 expression after neoadjuvant chemotherapy associates to lower survival in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-09-06.