Genetic Targeting of dSAMTOR, A Negative dTORC1 Regulator, during Drosophila Aging: A Tissue-Specific Pathology.

mTORC1 regulates mammalian cell metabolism and growth in response to diverse environmental stimuli. Nutrient signals control the localization of mTORC1 onto lysosome surface scaffolds that are critically implicated in its amino acid-dependent activation. Arginine, leucine and S-adenosyl-methionine (SAM) can serve as major mTORC1-signaling activators, with SAM binding to SAMTOR (SAM + TOR), a fundamental SAM sensor, preventing the protein's (SAMTOR's) inhibitory action(s) against mTORC1, thereby triggering its (mTORC1) kinase activity. Given the lack of knowledge regarding the role of SAMTOR in invertebrates, we have identified the Drosophila SAMTOR homologue (dSAMTOR) in silico and have, herein, genetically targeted it through the utilization of the GAL4/UAS transgenic tool. Survival profiles and negative geotaxis patterns were examined in both control and dSAMTOR-downregulated adult flies during aging. One of the two gene-targeted schemes resulted in lethal phenotypes, whereas the other one caused rather moderate pathologies in most tissues. The screening of head-specific kinase activities, via PamGene technology application, unveiled the significant upregulation of several kinases, including the dTORC1 characteristic substrate dp70S6K, in dSAMTOR-downregulated flies, thus strongly supporting the inhibitory dSAMTOR action(s) upon the dTORC1/dp70S6K signaling axis in Drosophila brain settings. Importantly, genetic targeting of the Drosophila BHMT bioinformatics counterpart (dBHMT), an enzyme that catabolizes betaine to produce methionine (the SAM precursor), led to severe compromises in terms of fly longevity, with glia-, motor neuron- and muscle-specific dBHMT downregulations exhibiting the strongest effects. Abnormalities in wing vein architectures were also detected in dBHMT-targeted flies, thereby justifying their notably reduced negative geotaxis capacities herein observed mainly in the brain-(mid)gut axis. In vivo adult fly exposure to clinically relevant doses of methionine revealed the mechanistic synergism of decreased dSAMTOR and increased methionine levels in pathogenic longevity, thus rendering (d)SAMTOR an important component in methionine-associated disorders, including homocystinuria(s).

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer.

Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. "Acetyl-chromatin" homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

Targeting of copper-trafficking chaperones causes gene-specific systemic pathology in Drosophila melanogaster: prospective expansion of mutational landscapes that regulate tumor resistance to cisplatin.

Copper, a transition metal, is an essential component for normal growth and development. It acts as a critical co-factor of many enzymes that play key roles in diverse cellular processes. The present study attempts to investigate the regulatory functions decisively controlling copper trafficking during development and aging of the Drosophila model system. Hence, through engagement of the GAL4/UAS genetic platform and RNAi technology, we herein examined the in vivo significance of Atox1 and CCS genes, products of which pivotally govern cellular copper trafficking in fly tissue pathophysiology. Specifically, we analyzed the systemic effects of their targeted downregulation on the eye, wing, neuronal cell populations and whole-body tissues of the fly. Our results reveal that, in contrast to the eye, suppression of their expression in the wing leads to a notable increase in the percentage of malformed organs observed. Furthermore, we show that Atox1 or CCS gene silencing in either neuronal or whole-body tissues can critically affect the viability and climbing capacity of transgenic flies, while their double-genetic targeting suggests a rather synergistic mode of action of the cognate protein products. Interestingly, pharmacological intervention with the anti-cancer drug cisplatin indicates the major contribution of CCS copper chaperone to cisplatin's cellular trafficking, and presumably to tumor resistance often acquired during chemotherapy. Altogether, it seems that Atox1 and CCS proteins serve as tissue/organ-specific principal regulators of physiological Drosophila development and aging, while their tissue-dependent downregulation can provide important insights for Atox1 and CCS potential exploitation as predictive gene biomarkers of cancer-cell chemotherapy responses.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. Hence, we show that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.