Talco estéril versus yodopovidona en pleurodesis como tratamiento del derrame pleural maligno. Estudio prospectivo no aleatorizado / Sterile talc versus povidone-iodine in pleurodesis as a treatment for malignant pleural effusion. Non-randomized prospective study

Comparar el uso del talco estéril versus yodopovidona como agentes químicos en pleurodesis para el tratamiento del derrame pleural maligno.Materiales y Métodos: Estudio clínico, analítico, observacional, prospectivo, el cual incluyó 12 pacientes con diagnóstico clínico e histopatológico de derrame pleural maligno, a quienes se les practicó pleurodesis empleando talco estéril y yodopovidona.Resultados : Edad promedio fue de 46,25 ± 17,3 y la neoplasia primaria más común fue el cáncer de mama, representando el 50% de los casos estudiados. El grupo de pacientes tratados con yodopovidona tuvo 100% de efectividad en la fusión pleural posterior a la pleurodesis, y presentaron menos complicaciones durante el procedimiento y 24 horas posteriores al mismo con respecto al grupo tratado con talco estéril, el cual tuvo un 71,4% de efectividad y un 28,6% de falla al procedimiento; asimismo, estos últimos presentaron mayor porcentaje de complicaciones. Por otra parte, no se evidenció recidiva del derrame pleural en los 30 días de valoración posteriores al procedimiento. Estas diferencias no fueron estadísticamente significativas.Conclusiones : Ambos agentes esclerosantes fueron eficaces para lograr la fusión de las pleuras en pacientes con derrame pleural maligno, siendo el talco estéril el agente con mayor tendencia a producir complicaciones y fallo del procedimiento, en comparación a la yodopovidona(AU)

To compare the use of sterile talc versus povidone-iodine as chemical agents on pleurodesis for the treatment of malignant pleural effusion.Materials and Methods : A total of 12 patients with clinical and histopathologic diagnose of malignant pleural effusion were enrolled in a clinical, analytic, observational and prospective trial, to whom sterile talc and povidone-iodine pleurodesis was applied.Results : The mean age was 46,25 ± 17,3 and the most common primary neoplasm was breast cancer, which was present in 50% of the surveyed cases. The group of patients who received povidone-iodine had 100% of effectiveness on post-pleurodesis pleural fusion, and had fewer complications during the procedure and 24 hours afterwards vis-à-vis the group who received sterile talc powder, which had 74.4% of effectiveness and 28.6% of procedure failure; furthermore, the last mentioned had higher percentage of complications. Moreover, there was no evidence of recurrence of pleural effusion in the 30 days post-procedure assessment.Conclusions : Both sclerosant agents were efficient to accomplish pleural fusion in patients with malignant pleural effusion, with sterile talc being the agent with higher tendency to generate more complications and procedure failure compared to povidone-iodine(AU)

Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation.

Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived (V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response.

TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response.

Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.

Vemurafenib resistance reprograms melanoma cells towards glutamine dependence.

BACKGROUND: (V600) BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells. METHODS: Isogenic BRAFi sensitive parental (V600) BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-L-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo. RESULTS: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells. CONCLUSION: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

The B55α subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation.

Glutamine is an essential nutrient for cancer cell survival and proliferation, yet the signaling pathways that sense glutamine levels remain uncharacterized. Here, we report that the protein phosphatase 2A (PP2A)-associated protein, α4, plays a conserved role in glutamine sensing. α4 promotes assembly of an adaptive PP2A complex containing the B55α regulatory subunit via providing the catalytic subunit upon glutamine deprivation. Moreover, B55α is specifically induced upon glutamine deprivation in a ROS-dependent manner to activate p53 and promote cell survival. B55α activates p53 through direct interaction and dephosphorylation of EDD, a negative regulator of p53. Importantly, the B55α-EDD-p53 pathway is essential for cancer cell survival and tumor growth under low glutamine conditions in vitro and in vivo. This study delineates a previously unidentified signaling pathway that senses glutamine levels as well as provides important evidence that protein phosphatase complexes are actively involved in signal transduction.

Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins.

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.

The BCL-2 protein BAK is required for long-chain ceramide generation during apoptosis.

The BCL-2 family members BAK and BAX are required for apoptosis and trigger mitochondrial outer membrane permeabilization (MOMP). Here we identify a MOMP-independent function of BAK as a required factor for long-chain ceramide production in response to pro-apoptotic stress. UV-C irradiation of wild-type (WT) cells increased long-chain ceramides; blocking ceramide generation prevented caspase activation and cell death, demonstrating that long-chain ceramides play a key role in UV-C-induced apoptosis. In contrast, UV-C irradiation did not increase long-chain ceramides in BAK and BAX double knock-out cells. Notably, this was not specific to the cell type (baby mouse kidney cells, hematopoietic) nor the apoptotic stimulus employed (UV-C, cisplatin, and growth factor withdrawal). Importantly, long-chain ceramide generation was dependent on the presence of BAK, but not BAX. However, ceramide generation was independent of the known downstream actions of BAK in apoptosis (MOMP or caspase activation), suggesting a novel role for BAK in apoptosis. Finally, enzymatic assays identified ceramide synthase as the mechanism by which BAK regulates ceramide metabolism. There was no change in CerS expression at the message or protein level, indicating regulation at the post-translational level. Moreover, CerS activity in BAK KO microsomes can be reactivated upon addition of BAK-containing microsomes. The data presented indicate that ceramide-induced apoptosis is dependent upon BAK and identify a novel role for BAK during apoptosis. By establishing a unique role for BAK in long-chain ceramide metabolism, these studies further demonstrate that the seemingly redundant proteins BAK and BAX have distinct mechanisms of action during apoptosis induction.

Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis.

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) delta inhibition. PKCdelta is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCdelta regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCdelta activity. Consistent with a potential role as a Rab7 activator, blocking PKCdelta function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCdelta inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.

Ceramide starves cells to death by downregulating nutrient transporter proteins.

Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast. These observations suggested to us that ceramide might kill mammalian cells by limiting cellular access to extracellular nutrients. Consistent with this proposal, physiologically relevant concentrations of ceramide produced a profound and specific downregulation of nutrient transporter proteins in mammalian cells. Blocking ceramide-induced nutrient transporter loss or supplementation with the cell-permeable nutrient, methyl pyruvate, reversed ceramide-dependent toxicity. Conversely, cells became more sensitive to ceramide when nutrient stress was increased by acutely limiting extracellular nutrients, inhibiting autophagy, or deleting AMP-activated protein kinase (AMPK). Observations that ceramide can trigger either apoptosis or caspase-independent cell death may be explained by this model. We found that methyl pyruvate (MP) also protected cells from ceramide-induced, nonapoptotic death consistent with the idea that severe bioenergetic stress was responsible. Taken together, these studies suggest that the cellular metabolic state is an important arbiter of the cellular response to ceramide. In fact, increasing nutrient demand by incubating cells in high levels of growth factor sensitized cells to ceramide. On the other hand, gradually adapting cells to tolerate low levels of extracellular nutrients completely blocked ceramide-induced death. In sum, these results support a model where ceramide kills cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation.