Loss of bmp15 function in the seasonal spawner Atlantic salmon results in ovulatory failure.

Bone morphogenetic protein 15 (BMP15) is an oocyte-specific growth factor important for successful female reproduction in mammals. While mutations in BMP15/Bmp15 cause ovulatory deficiency and/or infertility in certain mammalian species, loss of bmp15 in zebrafish, a continuous spawner and the only bmp15 knockout model in fish to date, results in complete arrest of follicle development and later female-to-male sex reversal, preventing to examine effects on ovulation/fertilization. Here, we used Atlantic salmon, a seasonal spawner, and generated bmp15 mutants to investigate ovarian development and fertility. Histological and morphometric analyses revealed that in biallelic frameshift (bmp15 fs/fs) mutant ovaries, folliculogenesis started earlier, resulting in an advanced development compared to wild-type (WT) controls, accompanied by a weaker expression of the (early) oocyte-specific factor figla. This precocious ovarian development was followed in bmp15 fs/fs females by enhanced follicle atresia during vitellogenic stages. Although genes involved in steroid synthesis and signaling (star, cyp11b, cyp17a1 and esr1) were dramatically higher in late vitellogenic bmp15 fs/fs mutant ovaries, estradiol-17ß plasma levels were lower than in WT counterparts, potentially reflecting compensatory changes at the level of ovarian gene expression. At spawning, bmp15 fs/fs females displayed lower gonado-somatic index values and reduced oocyte diameter, and the majority (71.4%), showed mature non-ovulating ovaries with a high degree of atresia. The remaining (28.6%) females spawned eggs but they either could not be fertilized or, upon fertilization, showed severe malformations and embryonic mortality. Our results show that Bmp15 is required for proper follicle recruitment and growth and later ovulatory success in Atlantic salmon, providing an alternative candidate target to induce sterility in farmed salmon. Moreover, since loss of bmp15 in salmon, in contrast to zebrafish, does not result in female-to-male sex change, this is the first mutant model in fish allowing further investigations on Bmp15-mediated functions in the ovulatory period.

Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors.

BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

Transcriptomic analysis of dead end knockout testis reveals germ cell and gonadal somatic factors in Atlantic salmon.

BACKGROUND: Sustainability challenges are currently hampering an increase in salmon production. Using sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently sterile salmon was produced by knocking out the germ cell-specific dead end (dnd). Several approaches may be applied to inhibit Dnd function, including gene knockout, knockdown or immunization. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes essential for the survival of gonadal somatic cells may be good alternative targets for sterility treatments. Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon. RESULTS: We have for the first time analysed RNA-sequencing data from germ cell-free (GCF)/dnd knockout and wild type (WT) salmon testis and searched for genes preferentially expressed in either germ cells or gonadal somatic cells. To exclude genes with extra-gonadal expression, our dataset was merged with available multi-tissue transcriptome data. We identified 389 gonad specific genes, of which 194 were preferentially expressed within germ cells, and 11 were confined to gonadal somatic cells. Interestingly, 5 of the 11 gonadal somatic transcripts represented genes encoding secreted TGF-ß factors; gsdf, inha, nodal and two bmp6-like genes, all representative vaccine targets. Of these, gsdf and inha had the highest transcript levels. Expression of gsdf and inha was further confirmed to be gonad specific, and their spatial expression was restricted to granulosa and Sertoli cells of the ovary and testis, respectively. Finally, we show that inha expression increases with puberty in both ovary and testis tissue, while gsdf expression does not change or decreases during puberty in ovary and testis tissue, respectively. CONCLUSIONS: This study contributes with transcriptome data on salmon testis tissue with and without germ cells. We provide a list of novel and known germ cell- and gonad somatic specific transcripts, and show that the expression of two highly active gonadal somatic secreted TGF-ß factors, gsdf and inha, are located within granulosa and Sertoli cells.

Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

A Novel Nanoprobe for Multimodal Imaging Is Effectively Incorporated into Human Melanoma Metastatic Cell Lines.

To facilitate efficient drug delivery to tumor tissue, several nanomaterials have been designed, with combined diagnostic and therapeutic properties. In this work, we carried out fundamental in vitro and in vivo experiments to assess the labeling efficacy of our novel theranostic nanoprobe, consisting of glycogen conjugated with a red fluorescent probe and gadolinium. Microscopy and resazurin viability assays were used to study cell labeling and cell viability in human metastatic melanoma cell lines. Fluorescence lifetime correlation spectroscopy (FLCS) was done to investigate nanoprobe stability. Magnetic resonance imaging (MRI) was performed to study T1 relaxivity in vitro, and contrast enhancement in a subcutaneous in vivo tumor model. Efficient cell labeling was demonstrated, while cell viability, cell migration, and cell growth was not affected. FLCS showed that the nanoprobe did not degrade in blood plasma. MRI demonstrated that down to 750 cells/µL of labeled cells in agar phantoms could be detected. In vivo MRI showed that contrast enhancement in tumors was comparable between Omniscan contrast agent and the nanoprobe. In conclusion, we demonstrate for the first time that a non-toxic glycogen-based nanoprobe may effectively visualize tumor cells and tissue, and, in future experiments, we will investigate its therapeutic potential by conjugating therapeutic compounds to the nanoprobe.

A co-culture model with brain tumor-specific bioluminescence demonstrates astrocyte-induced drug resistance in glioblastoma.

BACKGROUND: Although several studies suggest that stromal fibroblasts mediate treatment resistance in several cancer types, little is known about how tumor-associated astrocytes modulate the treatment response in brain tumors. Since traditionally used metabolic assays do not distinguish metabolic activity between stromal and tumor cells, and since 2-dimensional co-culture system does not recreate the formidable complexity of the microenvironment within 3-dimensional structures such as solid tumor tissue, we instead established a glioblastoma (GBM) cell-specific bioluminescent assay for direct measurements of tumor cell viability in the treatment of clinical relevant drugs. METHODS: Using lentiviral transfection, we established a panel of human GBM cell lines constitutively expressing a fusion transgene encoding luciferase and the enhanced green fluorescence protein (eGFP). We then initiated co-cultures with immortalized astrocytes, TNC-1, and the eGFP/Luc GBM cell lines. Next, we treated all eGFP/Luc GBM cell lines with Temozolomide (TMZ) or Doxorubicin, comparing co-cultures of glioblastoma (GBM) cells and TNC-1 astrocytes with mono-cultures of eGFP/Luc GBM cells. Cell viability was quantitated by measuring the luciferase expression. RESULTS: Titration experiments demonstrated that luciferase expression was proportional to the number of eGFP/Luc GBM cells, whereas it was not influenced by the number of TNC-1 cells present. Notably, the presence of TNC-1 astrocytes mediated significantly higher cell survival after TMZ treatment in the U251, C6, A172 cell lines as well as the in vivo propagated primary GBM tumor cell line (P3). Moreover, TNC-1 astrocytes mediated significantly higher survival after Doxorubicin treatment in the U251, and LN18 glioma cell lines. CONCLUSION: Glioma cell-specific bioluminescent assay is a reliable tool for assessment of cell viability in the brain tumor cell compartment following drug treatment. Moreover, we have applied this assay to demonstrate that astrocytes can modulate chemo sensitivity of GBM tumor cells. These effects varied both with the cell line and cytotoxic drug that were used, suggesting that several mechanisms may be involved.

In vitro treatment of melanoma brain metastasis by simultaneously targeting the MAPK and PI3K signaling pathways.

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAFV600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAFV600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

NUMB does not impair growth and differentiation status of experimental gliomas.

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide.

BACKGROUND: Expression of neuronal elements has been identified in various glial tumors, and glioblastomas (GBMs) with neuronal differentiation patterns have reportedly been associated with longer survival. However, the neuronal class III ß-tubulin has been linked to increasing malignancy in astrocytomas. Thus, the significance of neuronal markers in gliomas is not established. METHODS: The expressions of class III ß-tubulin, neurofilament protein (NFP), microtubule-associated protein 2 (MAP2) and neuron-specific enolase (NSE) were investigated in five GBM cell lines and two GBM biopsies with immunocytochemistry and Western blot. Moreover, the expression levels were quantified by real-time qPCR under different culture conditions. Following NSE siRNA treatment we used Electric cell-substrate impedance sensing (ECIS) to monitor cell growth and migration and MTS assays to study viability after irradiation and temozolomide treatment. Finally, we quantitated NSE expression in a series of human glioma biopsies with immunohistochemistry using a morphometry software, and collected survival data for the corresponding patients. The biopsies were then grouped according to expression in two halves which were compared by survival analysis. RESULTS: Immunocytochemistry and Western blotting showed that all markers except NFP were expressed both in GBM cell lines and biopsies. Notably, qPCR demonstrated that NSE was upregulated in cellular stress conditions, such as serum-starvation and hypoxia, while we found no uniform pattern for the other markers. NSE knockdown reduced the migration of glioma cells, sensitized them to hypoxia, radio- and chemotherapy. Furthermore, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than patients in the low-expression group. CONCLUSIONS: Neuronal markers are aberrantly expressed in human GBMs, and NSE is consistently upregulated in different cellular stress conditions. Knockdown of NSE reduces the migration of GBM cells and sensitizes them to hypoxia, radiotherapy and chemotherapy. In addition, GBM patients with high NSE expression had significantly shorter survival than patients with low NSE expression. Collectively, these data suggest a role for NSE in the adaption to cellular stress, such as during treatment.

Insulin-like 3 affects zebrafish spermatogenic cells directly and via Sertoli cells.

Pituitary hormones can use local signaling molecules to regulate target tissue functions. In adult zebrafish testes, follicle-stimulating hormone (Fsh) strongly increases the production of insulin-like 3 (Insl3), a Leydig cell-derived growth factor found in all vertebrates. Little information is available regarding Insl3 function in adult spermatogenesis. The Insl3 receptors Rxfp2a and 2b were expressed by type A spermatogonia and Sertoli and myoid cells, respectively, in zebrafish testis tissue. Loss of insl3 increased germ cell apoptosis in males starting at 9 months of age, but spermatogenesis appeared normal in fully fertile, younger adults. Insl3 changed the expression of 409 testicular genes. Among others, retinoic acid (RA) signaling was up- and peroxisome proliferator-activated receptor gamma (Pparg) signaling was down-regulated. Follow-up studies showed that RA and Pparg signaling mediated Insl3 effects, resulting in the increased production of differentiating spermatogonia. This suggests that Insl3 recruits two locally active nuclear receptor pathways to implement pituitary (Fsh) stimulation of spermatogenesis.

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was ß-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that ß-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of ß-sitosterol was linked to mitochondrial interference. Mechanistically, ß-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either ß-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, ß-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.

Divergent Activity of the Pseudogene PTENP1 in ER-Positive and Negative Breast Cancer.

Transcripts derived from the PTEN pseudogene (PTENP1) function as decoys to adsorb miRNAs targeting the PTEN tumor suppressor for degradation, and PTENP1 upregulation is known to inhibit growth in preclinical cancer models. Here, PTENP1 3'UTR transduction influences PTEN, AKT/mTOR signaling, and tumor progression in estrogen receptor (ER)-positive and -negative breast cancer cells. PTENP1 upregulation decreases PTEN gene expression in the ER-positive MCF7 and T47D human breast carcinoma cells and accelerates MCF7 tumor growth in vivo Of note, PTENP1 transduction significantly decreases ERα (ESR1) mRNA and protein levels in MCF7 xenografts with a concomitant increase in hsa-miR-26a, a miRNA known to target ESR1 In the ER-negative MDA-MB-231 and C3HBA breast cancer cells, upregulation of PTENP1 increases PTEN gene expression with no influence on hsa-miR-26a, ESR1, or ERα expression. While PTENP1 transduction did not influence the growth rate of human MDA-MB-231 xenografts, PTENP1 upregulation profoundly reduces its metastatic propensity. Furthermore, PTENP1 significantly inhibits the growth rate of ER-negative C3HBA murine breast cancer xenografts. PTENP1 transduction had no influence on doxorubicin cytotoxicity in ER-positive MCF7 cells but an increase in doxorubicin sensitivity was observed in the ER-negative MDA-MB-231 cells. In summary, while PTENP1 upregulation decreased PTEN transcript levels and stimulated the growth of ER-positive breast cancers, increased PTEN transcript levels and inhibited tumor progression was observed in the ER-negative cells.Implications: This report highlights the profound biological activity of PTENP1 in breast cancer, which is dictated by the hormone receptor status. Mol Cancer Res; 16(1); 78-89. ©2017 AACR.

Sex steroid production associated with puberty is absent in germ cell-free salmon.

In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gonadal steroidogenic system, subsequent development of secondary sex characteristics and reproductive behavior. To explore if the absence of germ cells affects puberty or growth in Atlantic salmon, germ cell-free (GCF), dnd knockout and wild type (WT) postsmolts were stimulated to enter puberty. No GCF fish entered puberty, whereas 66.7% (males) and 30% (females) WT fish completed or entered puberty, respectively. Expression of genes related to steroidogenesis (star, cyp17a1, cyp11ß, cyp19a1a), gonadal somatic cells (insl3, amh, igf3), oocytes (bmp15), gonadotropin receptors (fshr, lhcgr), and pituitary gonadotropic cells (fshb, lhb, gnrhr4) showed an immature status and failure to up-regulate gonadal sex steroid production in male and female GCF fish was also reflected in low or undetectable plasma sex steroids (11-ketotestosterone, estradiol-17ß and testosterone). A gender difference (high in females, low in males) was found in the expression of star and cyp17a1 in GCF fish. No clear difference in growth was detected between GCF and immature WT fish, while growth was compromised in maturing WT males. We demonstrate for the first time in a vertebrate that germ cells are required for pubertal activation of the somatic steroidogenic cells.

Antitumor efficacy improved by local delivery of species-specific endostatin.

OBJECT: Conflicting results have been reported concerning the antitumor efficacy of the angiogenesis inhibitor endostatin. This may be due to differences in the biological distribution of endostatin between studies or to the varying biological efficacies of the different protein forms that were examined. To address this issue, the authors used a local delivery approach in which each tumor cell secreted endostatin, providing uniform endostatin levels throughout the tumors. This allowed a direct assessment of the biological efficacy of soluble endostatin in vivo. METHODS: The authors genetically engineered BT4C gliosarcoma cells so that they would stably express and secrete either the human or murine form of endostatin. Endostatin-producing cells or mock-infected cells were implanted intracerebrally in syngeneic BD-IX rats. The antitumor efficacy of endostatin was evaluated on the basis of survival data and tumor volume comparisons. In addition, microvascular parameters were assessed. The authors confirmed the continuous release of endostatin by the BT4C cells. A magnetic resonance imaging-assisted comparison of tumor volumes revealed that local production of murine endostatin significantly inhibited tumor growth. Notably, 40% of the animals in this treatment group experienced long-term survival without histologically verifiable tumors 7 months after cell implantation. After local treatment with murine endostatin, tumor blood plasma volumes were reduced by 71%, microvessel density counts by 84%, and vascular area fractions by 75%. In contrast, human endostatin did not inhibit tumor growth significantly in this model. Centrally located regions of necrosis were present in tumors secreting both the human and the murine species-specific form of endostatin. CONCLUSIONS: The results suggest that endostatin inhibits tumor angiogenesis in vivo in a species-specific manner.

Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon.

Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.

Distribution patterns of the anti-angiogenic protein ADAMTS-1 during rat development.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) belongs to the large ADAM family of proteins. ADAMTS-1 contains a metalloproteinase domain, a disintegrin domain and three thrombospondin-like repeats but unlike ADAMs lacks a transmembrane domain. For the elucidation of the biological functions of ADAMTS-1, we raised new antibodies against ADAMTS-1. We show an accumulation of ADAMTS-1 protein at the basal lamina of rat embryonal epithelia of intestines, nasal cavity, choroid plexus, skin and in intracellular storage vesicles of epithelial cells. ADAMTS-1 protein seems to play a role in the development of the neuronal system, adipose tissue, muscle, heart, liver and adrenal glands. At the time of birth its presence is reduced in most organs. However, in the developing bone as well as in the skin, labelling increased towards late embryonal development. Immunoblots revealed a strong presence of a 62 kDa ADAMTS-1 fragment in kidneys, adrenal glands, lungs, intestines and heart. ADAMTS-1 was also present in the corresponding adult rat organs, but in more restricted distribution patterns. It was typically found in principal cells of collecting ducts, of the renal medulla, in ependymal cells of the ventricles and in some neurons. The results were confirmed by real-time PCR. The specific distribution pattern of ADAMTS-1 in a variety of organs during embryogenesis suggests a role of the molecule in tissue remodelling, vasculogenesis and angiogenesis.