Browsing by Subject "mTOR"

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  • Sanz, Dafne Jacome; Raivola, Juuli; Karvonen, Hanna; Arjama, Mariliina; Barker, Harlan; Murumägi, Astrid; Ungureanu, Daniela (2021)
    Simple Summary Ovarian cancer (OC) is known for its poor prognosis, due to the absence of reliable biomarkers and its late diagnosis, since the early-stage disease is almost asymptomatic. Lipid metabolism plays an important role in OC progression due to the development of omental metastasis in the abdominal cavity. The aim of our study was to assess the therapeutic role of various enzymes involved in lipid metabolism regulation or synthesis, in different subtypes of OC represented by cell lines as well as patient-derived cancer cell cultures (PDCs). We show that proprotein convertase subtilisin/kexin type 9 (PCSK9), a cholesterol-regulating enzyme, plays a pro-survival role in OC and targeting its expression impairs cancer cell growth. We also tested a small library of metabolic and mTOR-targeting drugs to identify drug vulnerabilities specific to various subtypes of OC. Our results show that in OC cell lines and PDCs the second generation of mTOR inhibitors such as AZD8055, vistusertib, dactolisib and sapanisertib, have higher cytotoxic activity compared to the first generation mTOR inhibitors such as rapalogs. These results suggest that, in the era of precision medicine, it is possible to target the metabolic pathway in OC and identify subtype-specific drug vulnerabilities that could be advanced to the clinic. Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic (n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures (PDCs, n = 5) to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements.
  • Olivieri, Fabiola; Ahtiainen, Maarit; Lazzarini, Raffaella; Pollanen, Eija; Capri, Miriam; Lorenzi, Maria; Fulgenzi, Gianluca; Albertini, Maria C.; Salvioli, Stefano; Alen, Markku J.; Kujala, Urho M.; Borghetti, Giulia; Babini, Lucia; Kaprio, Jaakko; Sipila, Sarianna; Franceschi, Claudio; Kovanen, Vuokko; Procopio, Antonio D. (2014)
    MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case-control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54-62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes' IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.
  • Barsby, Tom; Otonkoski, Timo (2022)
    The ability to maintain normoglycaemia, through glucose-sensitive insulin release, is a key aspect of postnatal beta cell function. However, terminally differentiated beta cell identity does not necessarily imply functional maturity. Beta cell maturation is therefore a continuation of beta cell development, albeit a process that occurs postnatally in mammals. Although many important features have been identified in the study of beta cell maturation, as of yet no unified mechanistic model of beta cell functional maturity exists. Here, we review recent findings about the underlying mechanisms of beta cell functional maturation. These findings include systemic hormonal and nutritional triggers that operate through energy-sensing machinery shifts within beta cells, resulting in primed metabolic states that allow for appropriate glucose trafficking and, ultimately, insulin release. We also draw attention to the expansive synergistic nature of these pathways and emphasise that beta cell maturation is dependent on overlapping regulatory and metabolic networks.
  • Arora, Amita; Kivelä, Annukka M.; Wang, Ling; Minkeviciene, Rimante; Taskinen, Juuso H.; Zhang, Birong; Koponen, Annika; Sun, Jing; Shirane, Michiko; Zhou, You; Hotulainen, Pirta; Raiborg, Camilla; Olkkonen, Vesa M. (2022)
    During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.
  • Liu, Ying; Mattila, Jaakko; Hietakangas, Ville (2020)
    Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. Here we report the results of an unbiased screen forDrosophilaTRs phosphorylated in an IIS-dependent manner. To conduct the screen, we built a library of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as theDrosophilaortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of thedPA2G4gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.