CytoCy5S™, a compound of many structures. in vitro and in vivo evaluation of four near-infrared fluorescent substrates of nitroreductase (NTR)

CytoCy5S™, a quenched, red-shifted fluorescent probe, has been used to exploit the imaging potential of the nitroreductase (NTR) reporter gene platform. Its use has been reported in a number of publications, however there are discrepancies in both the reported structure and its physicochemical properties. Herein, we aim to highlight these discrepancies and to define the best candidate of the four substrates under study for preclinical work in NTR reporting by optical applications. We report the synthesis, purification and characterisation of four NTR substrates, including alternately described structures currently referred by the name CytoCy5S. A comparative NTR enzymatic assay was performed to assess the spectroscopic characteristics of the different reductively activated probes. The NTR expressing triple-negative breast carcinoma cell line, MDA-MB-231 NTR+, was employed to compare, both in vitro and in vivo, the suitability of these fluorescent probes as reporters of NTR activity. Comparison of the reporting properties was achieved by flow cytometry, fluorescence microscopy and optical imaging, both in vivo and ex vivo. This study evaluated the different spectroscopic and biological characteristics of the four substrates and concluded that substrate 1 presents the best features for oncological in vivo preclinical optical imaging.publishedVersio

EVI1 as a Prognostic and Predictive Biomarker of Clear Cell Renal Cell Carcinoma

The transcription factor EVI1 plays an oncogenic role in several types of neoplasms by promoting aggressive cancer features. EVI1 contributes to epigenetic regulation and transcriptional control, and its overexpression has been associated with enhanced PI3K-AKT-mTOR signaling in some settings. These observations raise the possibility that EVI1 influences the prognosis and everolimus-based therapy outcome of clear cell renal cell carcinoma (ccRCC). Here, gene expression and protein immunohistochemical studies of ccRCC show that EVI1 overexpression is associated with advanced disease features and with poorer outcome-particularly in the CC-e.3 subtype defined by The Cancer Genome Atlas. Overexpression of an oncogenic EVI1 isoform in RCC cell lines confers substantial resistance to everolimus. The EVI1 rs1344555 genetic variant is associated with poorer survival and greater progression of metastatic ccRCC patients treated with everolimus. This study leads us to propose that evaluation of EVI1 protein or gene expression, and of EVI1 genetic variants may help improve estimates of prognosis and the benefit of everolimus-based therapy in ccRCC

Genetic Polymorphisms In Estrogen-related Genes And The Risk Of Breast Cancer Among Han Chinese Women

Exposure to high levels of estrogen is considered an important risk factor for susceptibility to breast cancer. Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk, but no comprehensive study has been performed among Han Chinese women. In the present study, 32 single-nucleotide polymorphisms (SNPs) in estrogen-related genes were genotyped using the MassARRAY IPLEX platform in 1076 Han Chinese women. Genotypic and allelic frequencies were compared between case and control groups. Unconditional logistic regression was used to assess the effects of SNPs on breast cancer risk. Associations were also evaluated for breast cancer subtypes stratified by estrogen receptor (ER) and progesterone receptor (PR) status. Case-control analysis showed a significant relation between heterozygous genotypes of rs700519 and rs2069522 and breast cancer risk (OR = 0.723, 95% CI = 0.541-0.965, p = 0.028 and OR = 1.500, 95% CI = 1.078-2.087, p = 0.016, respectively). Subgroup comparisons revealed that rs2446405 and rs17268974 were related to ER status, and rs130021 was associated with PR status. Our findings suggest that rs700519 and rs2069522 are associated with susceptibility to breast cancer among the Han Chinese population and have a cumulative effect with three other identified SNPs. Further genetic and functional studies are needed to identify additional SNPs, and to elucidate the underlying molecular mechanisms

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.publishedVersio

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis

Biomarcador; Histamina; LimfangioleiomiomatosiBiomarcador; Histamina; LinfangioleiomiomatosisBiomarker; Histamine; LymphangioleiomyomatosisInhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.This research was supported by AELAM, The LAM Foundation (Seed Grant 2019), Instituto de Salud Carlos III grants PI15/00854, PI18/01029, and ICI19/00047 (co-funded by European Regional Development Fund (ERDF), a way to build Europe), Generalitat de Catalunya SGR grants 2014-364 and 2017-449, the CERCA Program, and ZonMW-TopZorg grant 842002003. C.L.M. acknowledges the financial support (PRA-2017-51 project) of the University of Pisa. A.U.K. is supported by Nottingham Trent University’s Independent Fellowship Scheme

Brca1 Alternative Splicing Landscape In Breast Tissue Samples

Background: BRCA1 is a key protein in cell network, involved in DNA repair pathways and cell cycle. Recently, the ENIGMA consortium has reported a high number of alternative splicing (AS) events at this locus in blood-derived samples. However, BRCA1 splicing pattern in breast tissue samples is unknown. Here, we provide an accurate description of BRCA1 splicing events distribution in breast tissue samples. Methods: BRCA1 splicing events were scanned in 70 breast tumor samples, 4 breast samples from healthy individuals and in 72 blood-derived samples by capillary electrophoresis (capillary EP). Molecular subtype was identified in all tumor samples. Splicing events were considered predominant if their relative expression level was at least the 10% of the full-length reference signal. Results: 54 BRCA1 AS events were identified, 27 of them were annotated as predominant in at least one sample. Delta 5q, Delta 13, Delta 9, Delta 5 and del 1aA were significantly more frequently annotated as predominant in breast tumor samples than in blood-derived samples. Predominant splicing events were, on average, more frequent in tumor samples than in normal breast tissue samples (P = 0.010). Similarly, likely inactivating splicing events (PTC-NMDs, Non-Coding, Delta 5 and Delta 18) were more frequently annotated as predominant in tumor than in normal breast samples (P = 0.020), whereas there were no significant differences for other splicing events (No-Fs) frequency distribution between tumor and normal breast samples (P = 0.689). Conclusions: Our results complement recent findings by the ENIGMA consortium, demonstrating that BRCA1 AS, despite its tremendous complexity, is similar in breast and blood samples, with no evidences for tissue specific AS events. Further on, we conclude that somatic inactivation of BRCA1 through spliciogenic mutations is, at best, a rare mechanism in breast carcinogenesis, albeit our data detects an excess of likely inactivating AS events in breast tumor samples

Assessing Associations between the AURKA-HMMR-TPX2-TUBG1 Functional Module and Breast Cancer Risk in BRCA1/2 Mutation Carriers

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04-1.15, p = 1.9 x 10(-4) (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03-1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted pinteraction values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients' survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers

Tumor xenograft modeling identifies an association between TCF4 loss and breast cancer chemoresistance

Understanding the mechanisms of cancer therapeutic resistance is fundamental to improving cancer care. There is clear benefit from chemotherapy in different breast cancer settings; however, knowledge of the mutations and genes that mediate resistance is incomplete. In this study, by modeling chemoresistance in patientderived xenografts (PDXs), we show that adaptation to therapy is genetically complex and identify that loss of transcription factor 4 (TCF4; also known as ITF2) is associated with this process. A triple-negative BRCA1-mutaied PDX was used to study the genetics of chemoresistance. The PDX was treated in parallel with four chemotherapies for five iterative cycles. Exome sequencing identified few genes with de novo or enriched mutations in common among the different therapies, whereas many common depleted mutations/ genes were observed. Analysis of somatic mutations from The Cancer Genome Atlas (TCGA) supported the prognostic relevance of the identified genes. A mutation in TCF4 was found de novo in all treatments, and analysis of drug sensitivity profiles across cancer cell lines supported the link to chemoresistance. Loss of TCF4 conferred chemoresistance in breast cancer cell models, possibly by altering cell cycle regulation. Targeted sequencing in chemoresistant tumors identified an intronic variant of TCF4 that may represent an expression quantitative trait locus associated with relapse outcome in TCGA. Immunohistochemical studies suggest a common loss of nuclear TCF4 expression post-chemotherapy. Together, these results from tumor xenograft modeling depict a link between altered TCF4 expression and breast cancer chemoresistance

Decapping Protein Edc4 Regulates Dna Repair And Phenocopies Brca1

BRCA1 is a tumor suppressor that regulates DNA repair by homologous recombination. Germline mutations in BRCA1 are associated with increased risk of breast and ovarian cancer and BRCA1 deficient tumors are exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, uncovering additional components of this DNA repair pathway is of extreme importance for further understanding cancer development and therapeutic vulnerabilities. Here, we identify EDC4, a known component of processing-bodies and regulator of mRNA decapping, as a member of the BRCA1-BRIP1-TOPBP1 complex. EDC4 plays a key role in homologous recombination by stimulating end resection at double-strand breaks. EDC4 deficiency leads to genome instability and hypersensitivity to DNA interstrand cross-linking drugs and PARP inhibitors. Lack-of-function mutations in EDC4 were detected in BRCA1/2-mutation-negative breast cancer cases, suggesting a role in breast cancer susceptibility. Collectively, this study recognizes EDC4 with a dual role in decapping and DNA repair whose inactivation phenocopies BRCA1 deficiency