FGF-2 Gene Research Articles

Evaluation of pathologic and genomic characteristics in male breast cancer (MBC) patients.

1098 Background: MBC is a rare entity comprising less than 1% of breast cancers [Siegel RL 2017]. Due to the low incidence of MBC, information about the genomic landscape of MBC is lacking. Here we describe detailed pathologic and genomic characteristics of MBC patients. Methods: IRB approval was obtained for a retrospective analysis of archived pathology on patients treated at Cancer Treatment Centers of America. Comprehensive genomic profiling of tumors was derived from Foundation One next generation sequencing. Clinical information was derived from retrospective chart review. Inclusions: adult males with breast cancer with stage IV metastatic disease. Exclusions: Females, stage 0-IIIC disease, missing genomic and pathology information. Results of clinical, pathology and genomic data were summarized. Results: 10 patients met study criteria. Median age: 56 yrs., range 39-61 yrs. Race: 5/10 (50%) Caucasian, 5/10 (50%) African American. Number of prior treatment regimens: mean = 2.6 (range 0-6). Intrinsic subgroup: hormone receptor (HR)+/ HER2- 7/10 (70%), HR+/ HER2+ 2/10 (20%), HR-/ HER2+ 1/10 (10%), triple negative breast cancer (TNBC) 0/10 (0%). Tumor histology: invasive ductal carcinoma 10/10 (100%). Histology grade: poorly differentiated (diff.) 4/10 (40%), moderate diff. 4/10 (40%), well diff. 1/10 (10%), unknown 1/10 (10%). Biopsy site: primary tumor 4/10 (40%), metastatic site 4/10 (40%), liquid biopsy 1/10 (10%). Most frequent genomic alterations: PIK3CA 5/9 (56%), CCND1 4/9 (44%), ZNF703 3/9 (33%), FGF4 3/10 (33%), GATA3 3/9 (33%) FGF 19 3/9 (33%), FGF3 3/9 (33%) Alterations in FGF seen 14/38 (36.8%) of total genomic alterations. Most frequent alterations by primary tumor: ZNF703 3/4 (75%), FGF3 2/4 (50%), FGFR1 2/4 (50%), CCND1 2/4 (50%), FGF19 2/4 (50%), FGF4 2/4 (50%); by metastatic site: PIK3CA 4/5 (80%), GATA 3 2/5 (40%), CCND1 2/5 (40%), FGF3 1/5 (20%), FGF19 1/5 (20%), FGF4 1/5 (20%). Genomic alteration by histologic subgroup; HR+ HER2- PIK3CA 3/6 (50%), CCND1 3/6 (50%), FGF4 3/6 (50%), ZNF703 3/6 (50%), FGF19 3/6 (50%), FGF3 3/6 (50%); HR+/ HER2+: PIK3CA 2/2 (100%), CCND1 1/2 (50%), GATA3 1/2 (50%), MLL3 1/2 (50%); TNBC: BRCA2 1/1 (100%), BARD1 1/1 (100%). Microsatellite status was stable in 6/6 (100%) of patients. Conclusions: MBC patients display a heterogeneous variety of complex genomic alterations. Mutations in FGF genes were most commonly observed. Other common alterations seen in this series include PIK3CA, CCND1, ZNF703, and GATA3. Furthermore, the genomic profile of primary tumor site differed from the genomic profile of the metastatic site.

Mutational analysis using next-generation DNA sequencing in platinum-resistant muscle-invasive bladder cancer.

e16006 Background: Platinum-resistant muscle-invasive bladder cancer (prMIBC) is the main cause of bladder cancer mortality. Recent advances in next-generation DNA sequencing (NGS) has allowed characterization of the molecular landscape of MIBC. Yet, the correlation between the molecular alteration and the poor outcome of prMIBC is still lacking. This study aims to explore the potential genetic alterations in this group of patients. Methods: A retrospective study of prMIBC patients that had NGS molecular profiling at a single center from November 2014 to October 2018 was conducted. The prMIBC was defined as patients who had a significant residual disease or disease progression following cisplatin-based chemotherapy in a neoadjuvant or metastatic disease setting. NGS was performed on formalin-fixed paraffin-embedded tissue samples through commercial companies. Results: A total of 17 prMIBC with the median age of 65 [51-74] years old were included in this study. At the end of the study, 8 patients were alive and 9 deceased. A total of 58 mutations were identified. 7 of the 58 genes were mutated in ≥20% of cases: TP53 (65%), TERT promoter -124C > T (59%), ARID1A (35%), CDKN2A/B (30%), RB1 (24%), PIK3CA (24%), and FLCN (20%). Less frequent mutations (12% each) include MDM2, AKT2, KDM6A, SMARCA4, FGF3, FGF4, BRCA2, STAG2, and CCNE1 genes. Analysis of the mutations revealed key pathways including TP53/cell cycle pathway altered in 82%, PI-3K/mTOR pathway in 71%, Telomere regulation in 59%, SWI/SNF complex in 35% and histone modification in 24% of cases. Conclusions: Among patients with prMIBC, mutations in the p53/cell cycle pathway are the most common findings. Genetic alteration involving the PI-3K/mTOR pathway is frequently associated with prMIBC, which could provide an important lead for a predictive model and targeted intervention. Interestingly, the TERT promoter -124C > T mutation is a very common molecular alteration in prMIBC that has not been reported before. Telomere regulation may play a role in contributing to platinum-resistance in bladder cancer. Further elucidation of the molecular landscape in prMIBC on a larger scale would help to establish a molecular model to predict platinum-response in MIBC.

Fgf- and Bmp-signaling regulate gill regeneration in Ambystoma mexicanum

Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.

MiR-219a-5p enhances cisplatin sensitivity of human non-small cell lung cancer by targeting FGF9

Cisplatin (DDP) resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). MicroRNA-219a-5p (miR-219a-5p) has been reported to be a tumor suppressor in several cancers, but whether it regulates chemosensitivity in NSCLC remains unclear. Here, using quantitative real time PCR analysis, we observed that miR-219a-5p was down-regulated in responding tumor tissues compared with that in non-responding tumor tissues from NSCLC patients received DDP-based chemotherapy. Consistently, miR-219a-5p expression was lower in cisplatin (DDP)-resistant NSCLC cell lines (A549-R and SPC-A1-R) than that in corresponding parental cells (A549 and SPC-A1). Gain of-function assay showed ectopic expression of miR-219a-5p reversed DDP chemoresistance of NSCLC cells in vitro and in vivo. Using bioinformatics prediction and dual-luciferase reporter assays, we identified the FGF9 gene as a novel direct target of miR-219a-5p. Moreover, restoration of FGF9 expression reversed the miR-219a-5p-mediated chemosensitivity. In conclusion, this study demonstrated miR-219a-5p plays a crucial role in the development of acquired drug resistance to DDP in NSCLC cells by targeting FGF9 and might be a therapeutic target for DDP resistance in clinical practice.

Regulation of Proliferation, Differentiation and Functions of Osteoblasts by Runx2.

Runx2 is essential for osteoblast differentiation and chondrocyte maturation. During osteoblast differentiation, Runx2 is weakly expressed in uncommitted mesenchymal cells, and its expression is upregulated in preosteoblasts, reaches the maximal level in immature osteoblasts, and is down-regulated in mature osteoblasts. Runx2 enhances the proliferation of osteoblast progenitors by directly regulating Fgfr2 and Fgfr3. Runx2 enhances the proliferation of suture mesenchymal cells and induces their commitment into osteoblast lineage cells through the direct regulation of hedgehog (Ihh, Gli1, and Ptch1), Fgf (Fgfr2 and Fgfr3), Wnt (Tcf7, Wnt10b, and Wnt1), and Pthlh (Pthr1) signaling pathway genes, and Dlx5. Runx2 heterozygous mutation causes open fontanelle and sutures because more than half of the Runx2 gene dosage is required for the induction of these genes in suture mesenchymal cells. Runx2 regulates the proliferation of osteoblast progenitors and their differentiation into osteoblasts via reciprocal regulation with hedgehog, Fgf, Wnt, and Pthlh signaling molecules, and transcription factors, including Dlx5 and Sp7. Runx2 induces the expression of major bone matrix protein genes, including Col1a1, Spp1, Ibsp, Bglap2, and Fn1, in vitro. However, the functions of Runx2 in differentiated osteoblasts in the expression of these genes in vivo require further investigation.

Bioinformatics analysis of evolutionary characteristics and biochemical structure of FGF5 Gene in sheep

Fibroblast growth factor (FGF) 5 regulates the development and periodicity of hair follicles, which can affect hair traits. Loss-of-function mutations associated with long-hair phenotypes have been described in several mammalian species. Sheep is an important economic animal, however, the evolution characterizations and biological mechanism of oFGF5 (Ovis aries FGF5) gene are still poorly understood. In this study, oFGF5 gene was obtained by resequencing the whole genome of three Dorper sheep and RACE of two Kazakh sheep FGF5. We proposed FGF5 was phylogenetically related to FGF4 family and oFGF5 clearly orthologed to goat FGF5. Six loci were found from the positive selection results of FGF5 and half of them located on signal peptide. The basically similar rates of function-altering substitutions in sheep and goat lineage and the rest of the mammalian lineage of 365 SNPs indicated that the FGF5 gene was quite conservative during evolution. Homology modeling of the oFGF5 suggested that it has a highly conserved FGF superfamily domain containing 10 β-strands. Furthermore, the protein–protein docking analysis revealed that oFGF5 have the potential to form heterodimers with oFGFR1, the predicted interaction interface of FGF5-FGFR1 heterodimer was formed mainly by residues from FGF superfamily domain. Our observations suggested the evolutionary and structural biology features of oFGF5 might be relevant to its function about hair follicle development and modulating hair growth, and we confirmed our speculation by using the FGF5 gene editing sheep produced by CRISPR/Cas9 technology.

The Effect of miR-98 and miR-214 on Apoptotic and Angiogenic Pathways in Hepatocellular Carcinoma HepG2 Cells.

Hepatocellular carcinoma (HCC) is one of the foremost causes of cancer related morbidity worldwide. An increasing number of studies have confirmed that microRNAs play an important role in the development, progression and metastasis of HCC. From those important miRNAs are miR-98 and miR-214. This study were conducted to explore the effect of these two miRNAs on some apoptotic and angiogenic genes namely, BCL-2, survivin, CCND1, CDC2, P53 and P21, VEGF, Hif-1α, MMP-2, MMP-9, Ang-1, Ang-2, and FGF-1. miRNAs mimics and inhibitors transfection was used to investigate the role of both studied molecules in apoptosis and angiogenesis in HepG2 cells. QRT-PCR was used for Quantitative gene and miRNA expression analyses. The study revealed that miR-98 could serve as a pro-apoptotic factor through the upregulation of P53 gene expression levels. Besides, the anti-angiogenic effect of this miRNA was evident through the down regulation of Ang-1 and FGF-1 genes. Meanwhile, miR-214 showed a pro-apoptotic role and anti-angiogenic effects. These effects were verified through the significant down regulation of BCL-2, CDC2, VEGF, Ang-1 and MMP-2. These results introduced a possible positive role played by both miR-98 and miR-214 on some pro-apoptotic and anti-angiogenic genes.

Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes.

The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.

The sustained release of basic fibroblast growth factor accelerates angiogenesis and the engraftment of the inactivated dermis by high hydrostatic pressure.

We developed a novel skin regeneration therapy combining nevus tissue inactivated by high hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal autograft (CEA). The issue with this treatment is the unstable survival of CEA on the inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted subcutaneously onto the back of other mice and covered by CGS impregnated with saline or bFGF. Grafts were harvested after one, two and eight weeks, at which point the engraftment was evaluated through the histology and angiogenesis-related gene expressions were determined by real-time polymerase chain reaction. Histological sections showed that the dermal cellular density and newly formed capillaries in the bFGF group were significantly higher than in the control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF group was significantly higher than in the control group at Week 1. This study suggested that the angiogenesis into grafts was accelerated, which might improve the engraftment of inactivated dermis in combination with the sustained release of bFGF by CGSs.

Single nucleotide polymorphisms in MAP3K1, FGF10 and FGFR2 genes as breast cancer predictors

Introduction: Breast cancer (BC) is a heterogeneous disease caused by combination of several genetic, epigenetic and environmental factors. At about hundred single nucleotide polymorphisms significantly associate with the BC. Some of them are highly penetrant such as BRCA1 and BRCA2, but majority of them less penetrant.

Correction: HER2-Overexpressing Breast Cancers Amplify FGFR Signaling upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2+) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab.Experimental Design: HER2+ BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples.Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2+ early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2+ early breast cancer treated with neoadjuvant anti-HER2 therapy.Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR.

ER stress activation impairs the expression of circadian clock and clock-controlled genes in NIH3T3 cells via an ATF4-dependent mechanism

Endoplasmic reticulum (ER) stress and circadian clockwork signaling pathways mutually regulate various cellular functions, but the details regarding the cross-talk between these pathways in mammalian cells are unclear. In this study, whether perturbation of ER stress signaling affects the cellular circadian clockwork and transcription of clock-controlled genes was investigated in NIH3T3 mouse fibroblasts. An NIH3T3 cell model stably expressing luciferase (Luc) under the control of the Bmal1 clock gene promoter was established using a lentiviral system. Then, Luc activity was monitored in real-time to detect Bmal1-Luc oscillations. The ER stress activators thapsigargin (Tg) and tunicamycin (Tm) markedly reduced Bmal1-Luc oscillation amplitudes and induced phase delay shifts in NIH3T3 cells. Treatment with Tg/Tm activated ER stress signaling by upregulating GRP78, CHOP, ATF6, and ATF4 and simultaneously significantly decreased BMAL1 protein levels and inhibited the transcription of circadian clock (Bmal1, Per2, Nr1d1, and Dbp) and clock-controlled (Scad1, Fgf7, and Arnt) genes. 4-Phenylbutyric acid, an ER stress inhibitor, alleviated the transcriptional repression of the circadian clock genes and partially restored Bmal1-Luc oscillation amplitudes in Tg- or Tm-treated NIH3T3 cells. More importantly, knock-down of ATF4, but not ATF6, in Tg-treated NIH3T3 cells partially rescued Bmal1-Luc oscillation amplitudes and mRNA expression of the four circadian clock genes. Taken together, our study demonstrates that ER stress activation inhibits the transcription of circadian clock and clock-controlled genes via an ATF4-dependent mechanism.

Diversifying selection signatures among divergently selected subpopulations of Polish Red cattle

Polish Red cattle is one of the few indigenous breeds of European red cattle which is characterized by several desired features, such as high disease resistance, good health, longevity, good fertility, and high nutritional value of milk. Currently, Polish Red cattle population is a subject of two independent breeding programs: (i) improvement program and (ii) genetic resources conservation program. The aim of the improvement program is the genetic progress in terms of milk production and body conformation traits, while the conservation program mainly focuses on protection of the genetic resources of Polish Red cattle and preservation of the existing, original gene pool. By the analysis of FST genetic distances across genome-wide SNP panel, we detected diversifying selection signatures among these two subpopulations and indicated (among others) the significance of DGAT1 and FGF2 genes for milk production traits in these cattle. We also found that among genes being presumably under selection in terms of milk production, there are genes responsible, for example, for mammary gland development (e.g., SOSTDC1, PYGO2, MED1, and CCND1) and immune system response (e.g., IL10RA, IL12B, and IL21). The most important finding of this study is that the most pronounced genetic differences between the analyzed populations were associated with β-defensin genes (e.g., DEFB1, DEFB4A, DEFB5, DEFB7, DEFB10, DEFB13, EBD, BNBD-6, and LAP) located within so-called bovine cluster D on BTA27. The β-defensins are expressed mainly in the mammary gland and are antimicrobial peptides against the Gram-negative and Gram-positive bacteria, viruses, and other unicellular parasites. This suggests that antimicrobial resistance of mammary gland is of high importance during selection towards increased milk production and that genes responsible for this process are selected together with increasing levels of productivity.

Titanium dioxide nanotubes of defined diameter enhance mesenchymal stem cell proliferation via JNK- and ERK-dependent up-regulation of fibroblast growth factor-2 by T lymphocytes.

Long-term clinical success of a titanium implant not only depends upon osseointegration between implant and bone surface but also on the response of host immune cells. Following implantation of biomaterials, an inflammatory response, including T lymphocyte response, is ostensibly initiated by implant-cell interaction. However, little is known about the responses of T lymphocytes to titanium dioxide nanotubes. The present study aimed to explore the effect of titanium dioxide nanotubes on T lymphocytes in vitro and its biological consequences. The results of the present study showed that titanium dioxide nanotubes with diameter of 30-105 nm were non-cytotoxic to T lymphocytes, and the 105 nm titanium dioxide nanotube surface specifically possessed an ability to activate T lymphocytes, thus increasing DNA synthesis and cell proliferation. In addition, the 105 nm titanium dioxide nanotubes significantly activated the expression of FGF-2 gene and protein in T lymphocytes although smaller nanotubes (i.e. those with diameters of approximately 30 and 70 nm) had little effect on this. The present study investigated the mechanism by which 105 nm nanotubes stimulated FGF-2 expression in T lymphocytes by blocking key MAPK pathways. The inhibitors of JNK1/2/3 and ERK1/2 significantly inhibited 105 nm titanium dioxide nanotubes-induced FGF-2 expression. Corresponding to the increased expression of FGF-2, only the supernatant from T lymphocytes cultured on 105 nm nanotubes stimulated human mesenchymal stem cell proliferation. FGF-2 blocking antibody partially reversed the increased proliferation of human mesenchymal stem cells, supporting the role of T lymphocyte-derived FGF-2 in enhanced human mesenchymal stem cell proliferation. This suggests a significant role of T lymphocyte-titanium dioxide nanotube interaction in the proliferation of human mesenchymal stem cells, which is pivotal to the formation of new bone following implant placement.

Genome-wide differences in DNA methylation changes in caprine ovaries between oestrous and dioestrous phases

BackgroundDNA methylation plays a vital role in reproduction. Entire genome DNA methylation changes during the oestrous phase (ES) and dioestrous phase (DS) in the ovaries of Guanzhong dairy goats were investigated using bisulphite sequencing to understand the molecular biological mechanisms of these goats’ oestrous cycle.ResultsWe discovered distinct genome-wide DNA methylation patterns in ES and DS ovaries. A total of 26,910 differentially methylated regions were upregulated and 21,453 differentially methylated regions were downregulated in the ES samples compared with the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Differentially methylated region analysis showed hypomethylation in the gene body regions and hypermethylation in the joining region between upstream regions and gene bodies. The methylation ratios of the STAR, FGF2, FGF12, BMP5 and SMAD6 genes in the ES samples were lower than those of the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Conversely, the methylation ratios of the EGFR, TGFBR2, IGF2BP1 and MMD2 genes increased in the ES samples compared with the DS samples. In addition, 223 differentially methylated genes were found in the GnRH signalling pathway (KO04912), ovarian steroidogenesis pathway (KO04913), oestrogen signalling pathway (KO04915), oxytocin signalling pathway (KO04921), insulin secretion pathway (KO04911) and MAPK signalling pathway (KO04010).ConclusionsThis study is the first large-scale comparison of the high-resolution DNA methylation landscapes of oestrous and dioestrous ovaries from dairy goats. Previous studies and our investigations have shown that the NR5A2, STAR, FGF2 and BMP5 genes might have potential application value in regulating caprine oestrus.

Implementing a comprehensive translational oncology platform: from molecular testing to actionability

BackgroundIn order to establish the workflows required to implement a real-time process involving multi-omic analysis of patient samples to support precision-guided therapeutic intervention, a tissue acquisition and analysis trial was implemented. This report describes our findings to date, including the frequency with which mutational testing led to precision-guided therapy and outcome for those patients.MethodsEligible patients presenting to Oregon Health and Science University Knight Cancer Institute were enrolled on the study. Patients with biopsy proven metastatic or locally advanced unresectable prostate cancer, breast cancer, pancreatic adenocarcinoma, or refractory acute myelogenous leukemia receiving standard of care therapy were eligible. Metastatic site biopsies were collected and analyzed using the Knight Diagnostic Lab GeneTrails comprehensive solid tumor panel (124 genes). CLIA certified genomic information was made available to the treating physician.ResultsBetween 1/26/2017 and 5/30/2018, 38 patients were enrolled, with 28 successfully undergoing biopsy. Of these, 25 samples yielded sufficient tumor for analysis. The median biopsy cellularity and number of cores collected were 70% (15–90%) and 5 (2–20), respectively. No procedure-related complications occurred. GeneTrails analysis revealed that 22 of 25 (88%) tumor samples harbored at least one potentially actionable mutation, and 18 (72%) samples harbored 2 or more potentially actionable mutations. The most common genetic alterations identified involved: DNA damage repair genes, cell cycle regulating genes, PIK3CA/Akt/mTOR pathway, and FGF gene family. To date, CLIA certified genomic results were used by treating physicians for precision-guided therapy in 5 (23%) patients.ConclusionWe report the feasibility of real-time tissue acquisition and analysis to support a successful translational oncology platform. The workflow will provide the foundation to improve access and accrual to biomarker driven precision oncology trials.

Characterization of cis-regulatory elements for Fgf10 expression in the chick embryo.

Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018. © 2018 Wiley Periodicals, Inc.

Runx2 regulates cranial suture closure by inducing hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in suture mesenchymal cells.

Cleidocranial dysplasia (CCD, #119600), which is characterized by hypoplastic clavicles, open fontanelles, supernumerary teeth and a short stature, is caused by heterozygous mutations in RUNX2. However, it currently remains unclear why suture closure is severely impaired in CCD patients. The closure of posterior frontal (PF) and sagittal (SAG) sutures was completely interrupted in Runx2+/- mice, and the proliferation of suture mesenchymal cells and their condensation were less than those in wild-type mice. To elucidate the underlying molecular mechanisms, differentially expressed genes between wild-type and Runx2+/- PF and SAG sutures were identified by microarray and real-time reverse transcription polymerase chain reaction analyses. The expression of hedgehog, Fgf, Wnt and Pthlh signaling pathway genes, including Gli1, Ptch1, Ihh, Fgfr2, Fgfr3, Tcf7, Wnt10b and Pth1r, which were directly regulated by Runx2, was reduced in the sutures, but not the calvarial bone tissues of Runx2+/- mice. Bone formation and suture closure were enhanced in an organ culture of Runx2+/- calvariae with ligands or agonists of hedgehog, Fgf, Wnt and Pthlh signaling, while they were suppressed and suture mesenchymal cell proliferation was decreased in an organ culture of wild-type calvariae with their antagonists. These results indicate that more than a half dosage of Runx2 is required for the proliferation of suture mesenchymal cells, their condensation and commitment to osteoblast-lineage cells, and the induction of hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in sutures, but not in calvarial bone tissues, and also that the activation of hedgehog, Fgf, Wnt and Pthlh signaling pathways is necessary for suture closure.

FGF10 and the Mystery of Duodenal Atresia in Humans.

Background: Duodenal atresia (DA) is a congenital obstruction of the duodenum, which affects 1 in 7000 pregnancies and requires major surgery in the 1st days of life. Three morphological DA types are described. In humans, the association between DA and Down syndrome suggests an underlying, albeit elusive, genetic etiology. In mice, interruption of fibroblast growth factor 10 (Fgf10) gene signaling results in DA in 30–50% of embryos, supporting a genetic etiology. This study aims to validate the spectrum of DA in two novel strains of Fgf10 knock-out mice, in preparation for future and translational research.Methods: Two novel CRISPR Fgf10 knock-out mouse strains were derived and embryos generated by heterozygous plug-mating. E15.5–E19.5 embryos were genotyped with respect to Fgf10 and micro-dissected to determine the presence and type of DA.Results: One twenty seven embryos (32 wild-type, 34 heterozygous, 61 null) were analyzed. No wild-type or heterozygous embryos had DA. However, 74% of Fgf10 null embryos had DA (49% type 1, 18% type 2, and 33% type 3).Conclusion: Our CRISPR-derived strains showed higher penetrance of DA due to single-gene deletion of Fgf10 in mice than previously reported. Further, the DA type distribution in these mice more closely reiterated that observed in humans. Future experiments will document RNA and protein expression of FGF10 and its key downstream signaling targets in normal and atretic duodenum. This includes exploitation of modern, high-fidelity developmental tools, e.g., Fgf10flox/+–tomatoflox/flox mice.

Expression profiles and associations of FGF1 and FGF10 with intramuscular fat in Tibetan chicken

ABSTRACT1. FGF1 and FGF10, two paracrine members of the fibroblast growth factor (FGF) gene family, play critical roles in the development, structural and metabolic remodelling of adipose tissue.2. The objective of this study was to investigate the expression profiles of FGF1 and FGF10 genes in breast muscle and thigh muscle in 5 developmental stages (1, 81, 119, 154 and 210 d old) in Tibetan chickens. The possible relationships between expression of these genes and intramuscular fat (IMF) content were analysed in Tibetan chickens.3. Expression profiles showed that FGF1 and FGF10 mRNA were ubiquitously expressed in various tissues of 154-d-old Tibetan chickens. Lung tissue contained the highest amount of FGF1 and FGF10 mRNA while breast muscle and thigh muscle exhibited lower levels of FGF1 and FGF10 mRNA in both males and females compared with other tissues (P < 0.05). Temporal expression of FGF1 and FGF10 in breast and thigh muscle showed similar tendencies in males and females, respectively, with peaks in thigh muscle at 119-d-old and breast muscle in 1-d-old males and females, respectively.4. Correlation analysis suggested that gender had an influence on the relationships of FGF1 and FGF10 expression with IMF content in thigh muscle. The RNA levels of FGF1 and FGF10 genes in male thigh muscle were positively related to IMF content of Tibetan chickens (P < 0.01), while the correlations were shown to be negative in female thigh muscle (P > 0.05).5. These results provide a basis for functional elucidation of FGF1 and FGF10 genes on adipocyte development and intramuscular fat deposition, as well as selective breeding and resource exploration of local poultry breeds.