Loss of progesterone receptor is associated with distinct tyrosine kinase profiles in breast cancer.

PURPOSE: The aim of this study was to assess protein tyrosine kinase profiles in primary breast cancer samples in correlation with the distinct hormone and growth receptor profiles ER, PR, and HER2. EXPERIMENTAL DESIGN: Pamchip® microarrays were used to measure the phosphorylation of 144 tyrosine kinase substrates in 29 ER+ breast cancer samples and cell lines MCF7, BT474 and ZR75-1. mRNA expression data from the METABRIC cohort and publicly available PR chip-sequencing data were used for validation purposes, together with RT-PCR. RESULTS: In ER+ breast tumors and cell lines, we observed that the loss of PR expression correlated to higher kinase activity in samples and cell lines that were HER2-. A number of kinases, representing mostly proteins within the PI3K/AKT pathway, were identified as responsible for the differential phosphorylation between PR- and PR+ in ER+/HER2- tumors. We used the METABRIC cohort to analyze mRNA expression from 977 ER+/HER2- breast cancers. Twenty four kinase-encoding genes were identified as differentially expressed between PR+ and PR-, dividing ER+/HER2- samples in two distinct clusters with significant differences in survival (p < 0.05). Four kinase genes, LCK, FRK, FGFR4, and MST1R, were identified as potential direct targets of PR. CONCLUSIONS: Our results suggest that the PR status has a profound effect on tyrosine kinases, especially for FGFR4 and LCK genes, in ER+/HER2- breast cancer patients. The influence of these genes on the PI3K/AKT signaling pathway may potentially lead to novel drug targets for ER+/PR- breast cancer patients.

Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity.

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

Genome-wide mapping and analysis of aryl hydrocarbon receptor (AHR)- and aryl hydrocarbon receptor repressor (AHRR)-binding sites in human breast cancer cells.

The aryl hydrocarbon receptor (AHR) mediates the toxic actions of environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), and also plays roles in vascular development, the immune response, and cell cycle regulation. The AHR repressor (AHRR) is an AHR-regulated gene and a negative regulator of AHR; however, the mechanisms of AHRR-dependent repression of AHR are unclear. In this study, we compared the genome-wide binding profiles of AHR and AHRR in MCF-7 human breast cancer cells treated for 24 h with TCDD using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). We identified 3915 AHR- and 2811 AHRR-bound regions, of which 974 (35%) were common to both datasets. When these 24-h datasets were also compared with AHR-bound regions identified after 45 min of TCDD treatment, 67% (1884) of AHRR-bound regions overlapped with those of AHR. This analysis identified 994 unique AHRR-bound regions. AHRR-bound regions mapped closer to promoter regions when compared with AHR-bound regions. The AHRE was identified and overrepresented in AHR:AHRR-co-bound regions, AHR-only regions, and AHRR-only regions. Candidate unique AHR- and AHRR-bound regions were validated by ChIP-qPCR and their ability to regulate gene expression was confirmed by luciferase reporter gene assays. Overall, this study reveals that AHR and AHRR exhibit similar but also distinct genome-wide binding profiles, supporting the notion that AHRR is a context- and gene-specific repressor of AHR activity.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions.

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

Control of morphogenesis, protease secretion and gene expression in Candida albicans by the preferred nitrogen source ammonium.

Micro-organisms sense the availability of nutrients in their environment to control cellular behaviour and the expression of transporters and enzymes that are required for the utilization of these nutrients. In the pathogenic yeast Candida albicans, the preferred nitrogen source ammonium suppresses the switch from yeast to filamentous growth in response to certain stimuli, and it also represses the secretion of proteases, which are required for the utilization of proteins as an alternative nitrogen source. To investigate whether C. albicans senses the availability of ammonium in the extracellular environment or if ammonium uptake into the cell is required to regulate morphogenesis and gene expression, we compared the behaviour of wild-type cells and ammonium uptake-deficient mutants in the presence and absence of extracellular ammonium. Arginine-induced filamentous growth was suppressed by ammonium in the wild-type, but not in mutants lacking the ammonium permeases Mep1 and Mep2. Similarly, ammonium suppressed protease secretion and extracellular protein degradation in the wild-type, but not in mutants lacking the ammonium transporters. By comparing the gene expression profiles of C. albicans grown in the presence of low or high ammonium concentrations, we identified a set of genes whose expression is controlled by nitrogen availability. The repression of genes involved in the utilization of alternative nitrogen sources, which occurred under ammonium-replete conditions in the wild-type, was abrogated in mep1Δ mep2Δ mutants. These results demonstrate that C. albicans does not respond to the presence of sufficient amounts of the preferred nitrogen source ammonium by sensing its availability in the environment. Instead, ammonium has to be taken up into the cell to control morphogenesis, protease secretion and gene expression.

L-type CaV1.2 deletion in the cochlea but not in the brainstem reduces noise vulnerability: implication for CaV1.2-mediated control of cochlear BDNF expression.

Voltage-gated L-type Ca(2+) channels (L-VGCCs) like CaV1.2 are assumed to play a crucial role for controlling release of trophic peptides including brain-derived neurotrophic factor (BDNF). In the inner ear of the adult mouse, besides the well-described L-VGCC CaV1.3, CaV1.2 is also expressed. Due to lethality of constitutive CaV1.2 knock-out mice, the function of this ion channel as well as its putative relationship to BDNF in the auditory system is entirely elusive. We recently described that BDNF plays a differential role for inner hair cell (IHC) vesicles release in normal and traumatized condition. To elucidate a presumptive role of CaV1.2 during this process, two tissue-specific conditional mouse lines were generated. To distinguish the impact of CaV1.2 on the cochlea from that on feedback loops from higher auditory centers CaV1.2 was deleted, in one mouse line, under the Pax2 promoter (CaV1.2(Pax2)) leading to a deletion in the spiral ganglion neurons, dorsal cochlear nucleus, and inferior colliculus. In the second mouse line, the Egr2 promoter was used for deleting CaV1.2 (CaV1.2(Egr2)) in auditory brainstem nuclei. In both mouse lines, normal hearing threshold and equal number of IHC release sites were observed. We found a slight reduction of auditory brainstem response wave I amplitudes in the CaV1.2(Pax2) mice, but not in the CaV1.2(Egr2) mice. After noise exposure, CaV1.2(Pax2) mice had less-pronounced hearing loss that correlated with maintenance of ribbons in IHCs and less reduced activity in auditory nerve fibers, as well as in higher brain centers at supra-threshold sound stimulation. As reduced cochlear BDNF mRNA levels were found in CaV1.2(Pax2) mice, we suggest that a CaV1.2-dependent step may participate in triggering part of the beneficial and deteriorating effects of cochlear BDNF in intact systems and during noise exposure through a pathway that is independent of CaV1.2 function in efferent circuits.

Retrocochlear function of the peripheral deafness gene Cacna1d.

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination.

BACKGROUND: Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. FINDINGS: Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. CONCLUSIONS: The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Role of the Npr1 kinase in ammonium transport and signaling by the ammonium permease Mep2 in Candida albicans.

The ammonium permease Mep2 induces a switch from unicellular yeast to filamentous growth in response to nitrogen limitation in Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, the function of Mep2 and other ammonium permeases depends on the protein kinase Npr1. Mutants lacking NPR1 cannot grow on low concentrations of ammonium and do not filament under limiting nitrogen conditions. A G349C mutation in Mep2 renders the protein independent of Npr1 and results in increased ammonium transport and hyperfilamentous growth, suggesting that the signaling activity of Mep2 directly correlates with its ammonium transport activity. In this study, we investigated the role of Npr1 in ammonium transport and Mep2-mediated filamentation in C. albicans. We found that the two ammonium permeases Mep1 and Mep2 of C. albicans differ in their dependency on Npr1. While Mep1 could function well in the absence of the Npr1 kinase, ammonium transport by Mep2 was virtually abolished in npr1Δ mutants. However, the dependence of Mep2 activity on Npr1 was relieved at higher temperatures (37°C), and Mep2 could efficiently induce filamentous growth under limiting nitrogen conditions in npr1Δ mutants. Like in S. cerevisiae, mutation of the conserved glycine at position 343 in Mep2 of C. albicans to cysteine resulted in Npr1-independent ammonium uptake. In striking contrast, however, the mutation abolished the ability of Mep2 to induce filamentous growth both in the wild type and in npr1Δ mutants. Therefore, a mutation that improves ammonium transport by Mep2 under nonpermissible conditions eliminates its signaling activity in C. albicans.

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose.

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDP-glucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C. albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-d-galactose to alpha-d-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. Absence of C. albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevisiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C. albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose.