The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted ...much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells.
We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation.
We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment.
The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.
Abstract
Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug ...resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.
Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or ...sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.
Malignant melanoma is the most deadly form of skin cancer. There is a critical need to identify the patients that could be successfully treated by surgery alone and those that require adjuvant ...treatment. In this study, we demonstrate that the expression of tribbles2 (TRIB2) strongly correlates with both the presence and progression of melanocyte-derived malignancies. We examined the expression of TRIB2 in addition to 12 previously described melanoma biomarkers across three independent full genome microarray studies. TRIB2 expression was consistently and significantly increased in benign nevi and melanoma, and was highest in samples from patients with metastatic melanoma. The expression profiles for the 12 biomarkers were poorly conserved throughout these studies with only TYR, S100B and SPP1 showing consistently elevated expression in metastatic melanoma versus normal skin. Strikingly we confirmed these findings in 20 freshly obtained primary melanoma tissue samples from metastatic lesions where the expression of these biomarkers were evaluated revealing that TRIB2 expression correlated with disease stage and clinical prognosis. Our results suggest that TRIB2 is a meaningful biomarker reflecting diagnosis and progression of melanoma, as well as predicting clinical response to chemotherapy.
Cancer is one of the major causes of death worldwide, with Melanoma being the one of the ten most frequent malignancy in a number of countries. Melanoma is an extremely aggressive cancer and ...concomitant to this aggressiveness, patient prognosis is poor. As a result, novel therapies and cellular targets are desperately needed. Nowadays, the chemical compound BEZ235 has demonstrated significant potential as an anti-cancer agent. The PTEN/PI3K/AKT pathway constitutes an important signaling regulator of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. The PTEN is a dual protein/lipid phosphatase which most important substrate is the phosphatidyl-inositol-3,4,5-triphosphate (PIP3), the product of PI3K. An increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. TRIB2, a gene that has been reported to be up-regulated in some cancers, has also been implicated in the negative regulation of the FOXO signaling cascade, specifically the negative regulation of FOXO3a. Consequently TRIB2 has been implicated in Melanoma resistance to various classical chemotherapeutics, like DTIC, and to some PI3K inhibitors, like BEZ235. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research.BEZ235 is a potent inhibitor of PI3Ks that are constitutively active in many cancers, including Melanoma. This deregulation results in the inactivation of the FOXO family of transcription factors, critical regulators of the cell cycle and apoptosis. Here we investigate how TRIB2 mediates PI3K inhibitor resistance and the role(s) of FOXO3a in this response. Our finding implicate TRIB2 influencing apoptosis (although not the cell cycle) and that this occurs at the level of transcription. Our findings also indicate that the over expression of TRIB2 significantly attenuates BEZ235 induced apoptosis and confer resistance to p53-dependent chemotherapeutics that induce apoptosis. However, in contrast to BEZ235 exposure, we note that DTIC treatment stabilizes p53 in cells with an over expression of TRIB2. Our findings indicate that cellular balance between p53 and MDM2 is disrupted. Here we note also that TRIB2 transcription and protein expression is significantly higher in Melanoma patient samples compared to normal skin tissue.
Na infecção pelo HIV-1, importantíssimas células do sistema imunitário são infectadas, originando uma deterioração progressiva do sistema imunitário, o que o torna susceptível a infecções ...oportunistas. Este trabalho tem como objectivo perceber a forma como a célula hospedeira responde à infecção por HIV-1. A exposição dos seres eucariotas a diversas infecções virais ao longo do tempo modulou o sistema imunitário inato e adquirido, que evoluiu de forma a fazer face a essas infecções. Por outro lado, também os agentes infecciosos desenvolveram estratégias para escapar a essas defesas. Diversas células do sistema imunitário como macrófagos, células dendríticas, neutrófilos e células NK intervêm na resposta à patogénese viral. Para além destas células foram seleccionadas proteínas da própria célula que desempenham um papel fulcral na defesa da célula hospedeira contra a infecção pelo HIV-1, designadas por factores de restrição. Os factores abordados neste trabalho são: SAMHD1, TRIM5α, tetrina e APOBEC3G. Sendo a APOBEC3G a que detém maior actividade anti-HIV-1. A APOBEC3G tem como antagonista uma proteína viral acessória Vif. Na infecção pelo HIV-1 deficiente em Vif, o factor de restição APOBEC3G é incorporado nos viriões formados catalisando a desaminação de citidina do RNA viral. Induz assim hipermutações no DNA proviral e consequentemente, inibe a replicação viral em linhagens de células T não-permissivas para vírus deficientes em Vif. Por outro lado, na presença de Vif a proteína APOBEC3G é degradada antes da sua incorporação no virião, evitando a hipermutação do genoma viral. Vif pode inibir a acção de APOBEC3G por mecanismos distintos da indução da sua degradação como por exemplo inibindo a sua tradução ou competindo pela ligação a componentes virais. A interacção APOBEC3G-Vif é um bom alvo terapêutico para o desenvolvimento de novos medicamentos anti-retrovirais.
Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of ...hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia.
Considering the role of magnesium in bone metabolism and the increasing relevance of plant-mediated green-synthesis, this work compares the bone cytocompatibility of magnesium hydroxide nanoparticles ...(NPs) produced by using pure water, Mg(OH)2, or a rosehip (RH) aqueous extract, Mg(OH)2RH. The NPs were evaluated for dose- and time-dependent effects on human osteoblastic and osteoclastic response, due to the direct involvement of the two cell types in bone metabolism. Mg(OH)2 NPs presented nanoplatelet-like morphology (mean diameter ~90 nm) and a crystalline structure (XRD analysis); the RH-mediated synthesis yielded smaller rounded particles (mean diameter <10 nm) with decreased crystallinity. On the ATR–FTIR spectra, both NPs presented the characteristic Mg-OH peaks; Mg(OH)2RH exhibited additional vibration bands associated with the presence of phytochemicals. On osteoblastic cells, NPs did not affect cell growth and morphology but significantly increased alkaline phosphatase (ALP) activity; on osteoclastic cells, particles had little effect in protein content, tartrate-resistant acid phosphatase (TRAP) activity, percentage of multinucleated cells, and cell area. However, compared with Mg(OH)2, Mg(OH)2RH increased osteoblastic differentiation by inducing ALP activity and promoting the expression of Runx2, SP7, Col1a1, and ALP, and had a negative effect on the expression of the osteoclastic genes NFATC1, CA2, and CTSK. These observations suggest the potential usefulness of Mg(OH)2RH NPs in bone regeneration.
In this work, magnesium hydroxide NPs were synthesized using water (Mg(OH)
NPs) or a rose hip (RH) extract (Mg(OH)
RH NPs) and tested for the bone cells' effects in co-cultured osteoblastic and ...osteoclastic cells, using a Transwell
insert system, allowing reciprocal cell paracrine interactions. Behavior of each cell population was characterized for typical phenotype markers, at days 1 and 6. Cell cultures treated with osteogenic/osteoclastogenic inducers were used as positive control of cell differentiation. The NPs presented a round shape morphology with an average diameter ~90 nm (Mg(OH)
NPs) and below 10 nm (Mg(OH)
RH NPs. Both NPs induced osteoblastic and osteoclastic behavior similarly to that observed in induced osteoblastic and osteoclastic cultures (positive controls). Differences between the two types of particles were evident at the gene expression level. Compared to Mg(OH)
NPs, the green-synthesized NPs greatly increased the expression of osteoblastic genes coding for the early markers ALP and collagen type 1 and the later transcription factor osterix, while decreasing the expression of osteoclastogenic genes, namely the essential transcription factor NFATC1, TRAP and the genes coding for the functional markers CA2 and CTSK. Overall, a positive added effect could be hypothesized for Mg(OH)
RH NPs with potential usefulness to promote bone formation in regenerative applications.
Sarcopenia and sarcopenic obesity are currently considered major global threats for health and well-being. However, there is a lack of adequate preclinical models for their study. The present trial ...evaluated the suitability of aged swine by determining changes in adiposity, fatty acids composition, antioxidant status and lipid peroxidation, development of metabolic disturbances and structural changes in tissues and organs. Iberian sows with clinical evidence of aging-related sarcopenia were fed a standard diet fulfilling their maintenance requirements or an obesogenic diet for 100 days. Aging and sarcopenia were related to increased lipid accumulation and cellular dysfunction at both adipose tissue and non-adipose ectopic tissues (liver and pancreas). Obesity concomitant to sarcopenia aggravates the condition by increasing visceral adiposity and causing dyslipidemia, insulin resistance and lipotoxicity in non-adipose tissues. These results support that the Iberian swine model represents certain features of sarcopenia and sarcopenic obesity in humans, paving the way for future research on physiopathology of these conditions and possible therapeutic targets.
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