Mysterious sphingolipids: metabolic interrelationships at the center of pathophysiology

Metabolic pathways are complex and intertwined. Deficiencies in one or more enzymes in a given pathway are directly linked with genetic diseases, most of them having devastating manifestations. The metabolic pathways undertaken by sphingolipids are diverse and elaborate with ceramide species serving as the hubs of sphingolipid intermediary metabolism and function. Sphingolipids are bioactive lipids that serve a multitude of cellular functions. Being pleiotropic in function, deficiency or overproduction of certain sphingolipids is associated with many genetic and chronic diseases. In this up-to-date review article, we strive to gather recent scientific evidence about sphingolipid metabolism, its enzymes, and regulation. We shed light on the importance of sphingolipid metabolism in a variety of genetic diseases and in nervous and immune system ailments. This is a comprehensive review of the state of the field of sphingolipid biochemistry

Investigation of the clinical utility of two potential pro-oncogenic genes in prostate cancer and breast cancer

The Identification of novel and specific biomarkers is crucial to diagnosis, and prognosis, in patients with prostate and breast cancer. Because cancer therapies have side effects in patients, discovering and potentially targeting specific biomarkers could promote the use of personalised approach for a more effective treatment. Firstly, we have focused on the development of a monoclonal antibody-drug-based therapy, targeting prostate cancer stem cells (PCSCs), using a monoclonal antibody (mAb) previously generated in our laboratory against human endothelial protein C receptor (EPCR). PCSCs were isolated using lentivirus expressing the enhanced green fluorescent protein (EGFP) under NANOG-promoter generating two populations NANOG-EGFP+ and NANOG-EGFP- and analysed for EPCR expression. No significant difference was observed in the expression of EPCR between NANOG-EGFP+ and NANOGEGFP- cell populations. A lack of conclusive correlation was observed between EPCR deficient cells with epithelial-mesenchymal transition (EMT) markers, cancer stem cells (CSCs), and stem cell markers. Finally, Gene Expression Profiling Interactive Analysis (GEPIA) was used to look at the tissue expression in normal and tumour tissue, showing high expression of EPCR in endothelial cells. Finally, based tissue expression profiling, EPCR is not a suitable candidate for antibody targeting as it would lead to off-target effects in multiple tissues, therefore no further experiments were designed using EPCR as a target biomarker. Following this, a feasible study on the effect of Sperm-Associated Antigen 5 (SPAG5) chemoresistance and cancer progression in prostate and breast cancer was performed. The transcriptome and proteome of SPAG5 deficient were investigated in triple-negative breast cancer (TBC) MDA-MB-231 and androgenindependent prostate cancer DU145 cell lines, by RNA-sequencing and mass spectrometry (MS) analysis. Transcriptome was performed and a total of 2,201 differentially expressed genes (DEGs) in MDA-MB-231 SPAG5 deficient cells, while 907 DEGs DU145 SPAG5 deficient cells, versus control empty vector pLKO.1 cells, were identified. No significant differences in the cell cycle were observed in Doxorubicin and Epirubicin treatment DU145 and MDA-MB-231 SPAG5 deficient cells versus controls. A list of the most statistically significant genes upregulated and downregulated was taken forward for verification for common and unique pathways, through free available online resources such as METASCAPE, and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO). Using StatsPro free online sources proteomics analysis generated 230 differentially expressed proteins (DEPs) in MDA-MB- 231 SPAG5 deficient cells and 65 DEPs DU145 SPAG5 deficient versus control cells. Protein-protein interaction (PPI) network using Cytoscape has been conducted for enrichment KEGG analysis. Cross-over data from MS and RNAseq upregulated and downregulated genes in MDA-MB-231 and DU145 SPAG5 deficient were compared to in silico data from cBioPortal tool. Interestingly, positive correlation was observed in genes involved in cell cycle, but also in genes involved in catalyse and biosynthesis of cholesterol. Collectively those data offer a wider insight into the association of SPAG5 in cancer progression and its potential role not only in pathways involved in cell cycle but also how in lipid metabolism in cancer

Understanding the Role of Transforming Growth Factor Beta Signaling in the Uterus Using Genetically Modified Mouse Models

Transforming growth factor beta (TGFβ) superfamily signaling regulates multiple reproductive events. However, the in vivo role of the TGFβ signaling in uterine development and function is not well defined. Conditional knockout (cKO) of TGFβ receptor 1 (TGFBR1) in the female reproductive tract leads to remarkable smooth muscle defects. The first study of this dissertation research was to further define the role of TGFβ signaling in uterine development. We found that the myometrial defects in Tgfbr1 cKO mice were associated with dysregulated expression of key extracellular matrix components and platelet-derived growth factors signaling during a critical time window of early postnatal uterine development. To complement the loss of function model, in the second study, we generated a uterine specific gain-of-function mouse model harboring a constitutively active TGFBR1 which leads to over-activation of TGFβ signaling. Constitutive activation of TGFBR1 caused infertility and defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced numbers of uterine glands, and impaired uterine decidualization. These studies underscore the importance of a balanced TGFβ signaling system in establishing a uterine microenvironment conducive to normal development and function. In the third study of this dissertation research, we focused on identifying the role of TGFβ signaling in PTEN-inactivated uterine epithelial cells. Depletion of PTEN in the mouse uterus causes endometrial cancer. We found that simultaneous deletion of Tgfbr1 and Pten in the mouse uterus caused severe endometrial lesions and pulmonary metastases compared with deletion of Pten alone. The development of metastasis and accelerated tumor progression in the Pten/Tgfbr1 double knockout mice was linked to increased production of pro-inflammatory chemokines, enhanced cancer cell motility evidenced by myometrial invasion and disruption, and an altered tumor microenvironment characterized by recruitment of tumor-associated macrophages. Our results suggest that TGFβ signaling synergizes with PTEN to suppress the progression of endometrial cancer

Future Aspects of Tumor Suppressor Gene

Tumor suppressor genes (TSGs) and their signaling networks are fast growing areas in current biomedical science. These groups of genes, which are not limited to tumor suppression, play critical roles in many cellular activities. This book, "Future Aspects of Tumor Suppressor Genes", contains some fascinating fields, from basic to translational researches, in recent TSG studies. For example, several TSG signaling pathways are addressed in this book, and both mouse and Drosophila models used for the exploration of these genes are described based on the experimental evidence. A detailed review for current knowledge of microRNA studies in the regulation of tumor growth is introduced. Additionally, how natural compounds interfere with the progression of cancer development via TSG pathways is systemically summarized. Recent progresses in cell reprogramming and stemness transition processes regulated by TSG pathways are also included in this book

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Impact of Glycosyltransferases on the Phenotype, Signaling and Transcriptome of Colorectal Cancer Cell Lines. Focus on the role of glycosyltransferases B4GALNT2 and FUT6 and their cognate Sda and sLex antigens

In colorectal cancer (CRC), two carbohydrate structures are modulated: the Sda antigen, synthesized by B4GALNT2, and sLex antigen, mainly synthesized by FUT6. sLex antigen is often overexpressed and associated with worse prognosis; B4GALNT2/Sda antigen are dramatically downregulated but their role in tumor progression and development is not fully clear. TCGA interrogation revealed a dramatic down-regulation of B4GALNT2 mRNA in CRC, compared with normal samples. Patients with higher B4GALNT2 mRNA in CRC samples displayed longer survival. Yet, methylation and miRNA expression play a relevant role in B4GALNT2 downregulation in CRC. To clarify the mechanisms linking the B4GALNT2/Sda expression level to CRC phenotype, three different CRC cell lines were modified to express B4GALNT2: LS174T cell line, in which the constitutively expressed sLex antigen was partially replaced by Sda; SW480/SW620 pair, both lacking Sda and sLex antigens. In LS174T cells, the expression of B4GALNT2 reduced the ability to grow in poor adherence conditions and the expression of ALDH, a stemness marker. In SW620 cells, B4GALNT2 expression impacted on the main aspects of malignancy. In SW480 cells the expression of B4GALNT2 left unchanged the proliferation rate and the wound healing ability. To clarify the impact of sLex on CRC phenotype, the SW480/SW620 pair were permanently transfected to express FUT6 cDNA. In both cell lines, overexpression of FUT6/sLex boosted the clonogenic ability in standard growth conditions. Conversely, the growth in soft agar and the capacity to close a wound were enhanced only in SW620 cells. Transcriptome analysis of CRC cell lines transfected either with B4GALNT2 or FUT6 showed a relevant impact of both enzymes on gene expression modulation. Overall, current data may help to personalize therapies for CRC patients according to the B4GALNT2 levels and support a causal effect of this glycosyltransferase on reducing malignancy independently of sLex inhibition