FGFR2 risk SNPs confer breast cancer risk by augmenting oestrogen responsiveness.

The fibroblast growth factor receptor 2 (FGFR2) locus is consistently the top hit in genome-wide association studies for oestrogen receptor-positive (ER(+)) breast cancer. Yet, its mode of action continues to be controversial. Here, we employ a systems biology approach to demonstrate that signalling via FGFR2 counteracts cell activation by oestrogen. In the presence of oestrogen, the oestrogen receptor (ESR1) regulon (set of ESR1 target genes) is in an active state. However, signalling by FGFR2 is able to reverse the activity of the ESR1 regulon. This effect is seen in multiple distinct FGFR2 signalling model systems, across multiple cells lines and is dependent on the presence of FGFR2. Increased oestrogen exposure has long been associated with an increased risk of breast cancer. We therefore hypothesized that risk variants should reduce FGFR2 expression and subsequent signalling. Indeed, transient transfection experiments assaying the three independent variants of the FGFR2 risk locus (rs2981578, rs35054928 and rs45631563) in their normal chromosomal context show that these single-nucleotide polymorphisms (SNPs) map to transcriptional silencer elements and that, compared with wild type, the risk alleles augment silencer activity. The presence of risk variants results in lower FGFR2 expression and increased oestrogen responsiveness. We thus propose a molecular mechanism by which FGFR2 can confer increased breast cancer risk that is consistent with oestrogen exposure as a major driver of breast cancer risk. Our findings may have implications for the clinical use of FGFR2 inhibitors.Breast Cancer Research FoundationThis is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/carcin/bgw06

Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in gastrointestinal tumour

The human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumour-suppressor gene inactivated by methylation in several cancers. In this study, we analysed the methylation and expression status of hDAB2IP in gastrointestinal tumours. The promoter region of hDAB2IP was divided into two regions (m2a and m2b) based on our previous report, and the methylation status was determined by bisulphite DNA sequencing in gastric cancer cell lines. The gene expression was semiquantified by real-time RT–PCR, and the results indicated that the m2b promoter region might be an authentic methylation-mediated key regulator of the gene expression. Based on the sequence data, we developed a methylation-specific PCR (MSP) for the m2a and m2b regions and applied it to the samples. Methylation-specific PCR revealed aberrant methylation in the m2a region in eight of 12 gastric cancer cell lines (67%), 16 of 35 gastric cancer tissues (46%) and 29 of 60 colorectal cancer tissues (48%), and in the m2b region in eight of 12 cell lines (67%), 15 of 35 gastric cancer tissues (43%) and 28 of 60 colorectal cancer tissues (47%). On the other hand, seven (12%) and 11 (19%) of 59 gastrointestinal nonmalignant mucosal specimens showed methylation in the m2a and m2b regions, respectively, suggesting that hDAB2IP methylation might play a causative role in carcinogenesis. The 5-aza-2′-deoxycytidine treatment restored the gene expression in the m2b-methylated cell lines, confirming that the methylation caused gene downregulation. We also examined the relationship between hDAB2IP methylation and the clinicopathological features in patients with primary tumours, and determined that methylation in the m2b region was associated with location of the tumour in the stomach. In summary, our results demonstrated that hDAB2IP methylation is frequently present in gastrointestinal tumours and that the resulting gene silencing plays an important role in gastrointestinal carcinogenesis

Polarization characteristics of phase retardation defect mode lasing in polymeric cholesteric liquid crystals

We have studied the lasing characteristics of a dye-doped nematic layer sandwiched by two polymeric cholesteric liquid crystal (CLC) films as photonic band gap (PBG) materials. The nematic layer acts as a defect layer, the anisotropy of which brings about the following remarkable optical characteristics: (1) reflectance in the PBG region exceeds 50% due to the retardation effect, being unpredictable from a single CLC film; (2) efficient lasing occurs either at the defect mode wavelength or at the photonic band edge; and (3) the lasing emission due to both the defect mode and the photonic band edge mode contains both right- and left-circular polarizations, while the lasing emission from a dye-doped single CLC layer with a left-handed helix is left-circularly polarized.open2

DNA methylation status of REIC/Dkk-3 gene in human malignancies

The REIC (reduced expression in immortalized cells)/Dkk-3 is down-regulated in various cancers and considered to be a tumor suppressor gene. REIC/Dkk-3 mRNA has two isoforms (type-a,b). REIC type-a mRNA has shown to be a major transcript in various cancer cells, and its promoter activity was much stronger than that of type-b. In this study, we examined the methylation status of REIC/Dkk-3 type-a in a broad range of human malignancies. We examined REIC/Dkk-3 type-a methylation in breast cancers, non-small-cell lung cancers, gastric cancers, colorectal cancers, and malignant pleural mesotheliomas using a quantitative combined bisulfite restriction analysis assay and bisulfate sequencing. REIC/Dkk-3 type-a and type-b expression was examined using reverse transcriptional PCR. The relationships between the methylation and clinicopathological factors were analyzed. The rate of REIC/Dkk-3 type-a methylation ranged from 26.2 to 50.0% in the various primary tumors that were examined. REIC/Dkk-3 type-a methylation in breast cancer cells was significantly heavier than that in the other cell lines that we tested. REIC/Dkk-3 type-a methylation was inversely correlated with REIC/Dkk-3 type-a expression. There was a correlation between REIC/Dkk-3 type-a and type-b mRNA expression. REIC/Dkk-3 type-a expression was restored in MDA-MB-231 cells using 5-aza-2'-deoxycytidine treatment. We found that estrogen receptor-positive breast cancers were significantly more common among the methylated group than among the non-methylated group. REIC/Dkk-3 type-a methylation was frequently detected in a broad range of cancers and appeared to play a key role in silencing REIC/Dkk-3 type-a expression in these malignancies

The Gonium pectorale genome demonstrates co-option of cell cycle regulation during the evolution of multicellularity

Citation: Hanschen, E. R., Marriage, T. N., Ferris, P. J., Hamaji, T., Toyoda, A., Fujiyama, A., . . . Olson, B. (2016). The Gonium pectorale genome demonstrates co-option of cell cycle regulation during the evolution of multicellularity. Nature Communications, 7, 10. doi:10.1038/ncomms11370Additional Authors: Anderson, J.;Bakaric, R.;Luria, V.;Karger, A.;Kirschner, M. W.;Durand, P. M.;Michod, R. E.;Nozaki, H.The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions

Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

<p>Abstract</p> <p>Background</p> <p>Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the <it>ras-association domain isoform A (RASSF1A) </it>and the <it>death-associated protein kinase (DAPK) </it>genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-<it>ras</it>, <it>p53</it>, and <it>EGFR </it>genes determined previously on these same lung tumors.</p> <p>Methods</p> <p>Promoter methylation of the <it>RASSF1A </it>and <it>DAPK </it>genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-<it>ras</it>, <it>p53</it>, and <it>EGFR </it>were obtained from our previous studies.</p> <p>Results</p> <p>The <it>RASSF1A </it>gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The <it>DAPK </it>gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the <it>RASSF1A </it>or <it>DAPK </it>genes did not correlate with the frequency of mutations of the K<it>-ras, p53</it>, and <it>EGFR </it>gene.</p> <p>Conclusion</p> <p>Our results showed that <it>RASSF1A </it>and <it>DAPK </it>genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-<it>ras</it>, <it>p53 </it>and <it>EGFR </it>genes, suggesting each of these events may represent independent event in non-small lung tumorigenesis.</p

Single Cell Analysis Facilitates Staging of Blimp1-Dependent Primordial Germ Cells Derived from Mouse Embryonic Stem Cells

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKitbright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKitbright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5–10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKitbright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development

Primordial Germ Cell Specification from Embryonic Stem Cells

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Methodology and Principal Findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stellanegative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation