ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

Disruption of male fertility-critical Dcaf17 dysregulates mouse testis transcriptome.

During mammalian spermatogenesis, the ubiquitin proteasome system maintains protein homoeostasis (proteastasis) and spermatogenic cellular functions. DCAF17 is a substrate receptor in the ubiquitin CRL4 E3 Ligase complex, absence of which causes oligoasthenoteratozoospermia in mice resulting in male infertility. To determine the molecular phenomenon underlying the infertility phenotype caused by disrupting Dcaf17, we performed RNA-sequencing-based gene expression profiling of 3-weeks and 8-weeks old Dcaf17 wild type and Dcaf17 disrupted mutant mice testes. At three weeks, 44% and 56% differentially expressed genes (DEGs) were up- and down-regulated, respectively, with 32% and 68% DEGs were up- and down-regulated, respectively at 8 weeks. DEGs include protein coding genes and lncRNAs distributed across all autosomes and the X chromosome. Gene ontology analysis revealed major biological processes including proteolysis, regulation of transcription and chromatin remodelling are affected due to Dcaf17 disruption. We found that Dcaf17 disruption up-regulated several somatic genes, while germline-associated genes were down-regulated. Up to 10% of upregulated, and 12% of downregulated, genes were implicated in male reproductive phenotypes. Moreover, a large proportion of the up-regulated genes were highly expressed in spermatogonia and spermatocytes, while the majority of downregulated genes were predominantly expressed in round spermatids. Collectively, these data show that the Dcaf17 disruption affects directly or indirectly testicular proteastasis and transcriptional signature in mouse.

Fascin is essential for mammary gland lactogenesis.

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin-/- dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin-/- mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin-/- epithelial fraction was significantly reduced. Further in-depth analyses of fascin-/- mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and ß-casein in fascin-/- mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains.

Sperm-specific phospholipase C zeta (PLCζ) initiates intracellular calcium (Ca2+) transients which drive a series of concurrent events collectively termed oocyte activation. Numerous investigations have linked abrogation and absence/reduction of PLCζ with forms of male infertility in humans where oocyte activation fails. However, very few studies have examined potential relationships between PLCζ and advancing male age, both of which are increasingly considered to be major effectors of male fertility. Initial efforts in humans may be hindered by inherent PLCζ variability within the human population, alongside a lack of sufficient controllable repeats. Herein, utilizing immunoblotting, immunofluorescence, and quantitative reverse transcription PCR (qRT-PCR) we examined for the first time PLCζ protein levels and localization patterns in sperm, and PLCζ mRNA levels within testes, from mice at 8 weeks, 12 weeks, 24 weeks, and 36 weeks of age, from two separate strains of mice, C57BL/6 (B6; inbred) and CD1 (outbred). Collectively, advancing male age generally diminished levels and variability of PLCζ protein and mRNA in sperm and testes, respectively, when both strains were examined. Furthermore, advancing male age altered the predominant pattern of PLCζ localization in mouse sperm, with younger mice exhibiting predominantly post-acrosomal, and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ. However, the specific pattern of such decline in levels of protein and mRNA was strain-specific. Collectively, our results demonstrate a negative relationship between advancing male age and PLCζ levels and localization patterns, indicating that aging male mice from different strains may serve as useful models to investigate PLCζ in cases of male infertility and subfertility in humans.

Expression profiling of WD40 family genes including DDB1- and CUL4- associated factor (DCAF) genes in mice and human suggests important regulatory roles in testicular development and spermatogenesis.

BACKGROUND: The WD40-repeat containing proteins, including DDB1-CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development. RESULTS: Publically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60-73% of the genes were highly or specifically expressed in testis. Similarly, around 74-83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes. CONCLUSIONS: We have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

Estimating transfection efficiency in differentiated and undifferentiated neural cells.

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.

Vasculature reconstruction of decellularized liver scaffolds via gelatin-based re-endothelialization.

Decellularized liver scaffolds based liver engineering is a promising approach toward developing functional liver surrogates. However, a major obstacle to long-term transplantation is the hemocompatibility of the bioengineered liver surrogates. One approach to improve the hemocompatibility of engineered liver surrogates is re-endothelialization. In the current study, immortalized endothelial cells were perfused for re-endothelialization of decellularized rat liver scaffolds. When compared to the media-based perfusion approach, gelatin hydrogels-based perfusion significantly increased the number of cells that were retained in the decellularized liver scaffolds and the vascular lumen coverage ratio. Endothelial cells were lining along the vasculatures of the decellularized liver scaffolds and actively proliferating. Re-endothelialization improved the blood retention ability of the liver scaffold vasculatures. Doppler ultrasound detected active blood flows within the re-endothelialized liver scaffold transplants 8 days post-transplantation. Our results strengthened the feasibility of developing bioengineered liver surrogates utilizing decellularized liver scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 392-402, 2019.

Penile autotransplantation in rats: An animal model.

CONTEXT: Penile allotransplantation might be a viable option for patients who need penile reconstruction. AIMS: A successful autotransplantation rat model is the first step toward proceeding for allotransplantation. We herein evaluate autotransplantation following transaction of the rat penis just distal to the urethral bulb. SETTINGS AND DESIGN: Experimental animal study. MATERIALS AND METHODS: Five Sprague-Dawely rats weighing 520 g (SD 19) were used. Utilizing a magnification of 6-40, transection and immediate anastomosis of the tunica albuginea, urethra, dorsal vein and nerves were carried out. Vesicostomy was made to divert urine. The glandular skin was sutured to the perineum and the abdominal wall was closed in layers. STATISTICAL ANALYSIS USED: Descriptive statistics. RESULTS: Average surgery time was 8 h. The first two rats had no vesicostomy and died in the first postoperative day from retention. Three rats tolerated well the procedure and survived to the end point. One rat was sacrificed at day 10 and histopathology showed 30-50% necrosis of the implanted penis. Another rat was sacrificed at day 20 and showed normal cavernous tissue. The fifth rat was sacrificed 3 months postoperatively and showed evidence of moderate corporal fibrosis. Urethral fistula and necrosis of corpus spongiosum, dorsal nerve necrosis and dorsal vein occurred in all animals. CONCLUSIONS: Penile autotransplantation in rats is feasible and provides the basis for evaluation of the corpora cavernosa in an allotransplantation model. Long-term urethral continuity and dorsal neurovascular bundle survival in this model is difficult to establish.