Role of ABCG1 and ABCA1 in regulation of neuronal cholesterol efflux to apolipoprotein E discs and suppression of amyloid-β peptide generation

Maintenance of an adequate supply of cholesterol is important for neuronal function, whereas excess cholesterol promotes amyloid precursor protein (APP) cleavage generating toxic amyloid-β (Aβ) peptides. To gain insights into the pathways that regulate neuronal cholesterol level, we investigated the potential for reconstituted apolipoprotein E (apoE) discs, resembling nascent lipoprotein complexes in the central nervous system, to stimulate neuronal [3H]cholesterol efflux. ApoE discs potently accelerated cholesterol efflux from primary human neurons and cell lines. The process was saturable (17.5 μg of apoE/ml) and was not influenced by APOE genotype. High performance liquid chromatography analysis of cholesterol and cholesterol metabolites effluxed from neurons indicated that <25% of the released cholesterol was modified to polar products (e.g. 24-hydroxycholesterol) that diffuse from neuronal membranes. Thus, most cholesterol (∼75%) appeared to be effluxed from neurons in a native state via a transporter pathway. ATP-binding cassette transporters ABCA1, ABCA2, and ABCG1 were detected in neurons and neuroblastoma cell lines and expression of these cDNAs revealed that ABCA1 and ABCG1 stimulated cholesterol efflux to apoE discs. In addition, ABCA1 and ABCG1 expression in Chinese hamster ovary cells that stably express human APP significantly reduced Aβ generation, whereas ABCA2 did not modulate either cholesterol efflux or Aβ generation. These data indicate that ABCA1 and ABCG1 play a significant role in the regulation of neuronal cholesterol efflux to apoE discs and in suppression of APP processing to generate Aβ peptides

Ex vivo assessment of targeted therapies in a rare metastatic epithelial–myoepithelial carcinoma

Epithelial–myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial–myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial–myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC

CDK‐mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC‐driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer

Targeting SOX9 for degradation to inhibit chemoresistance, metastatic spread, and recurrence

Cancer cells with stem-like properties are believed to contribute to treatment resistance, dissemination, and recurrence. SOX9 controls stem cell plasticity and its deregulation may provide a basis for tumor progression. Here, we summarize our findings of targeted SOX9 destruction by SCFFBW7 (Skp1/Cul1/F-box) in medulloblastoma and its potential for therapeutic intervention

Inhibition of lysosomal function in macrophages incubated with elevated glucose concentrations:A potential contributory factor in diabetes-associated atherosclerosis

Objective: People with diabetes have an elevated risk of atherosclerosis. The accumulation of lipid within macrophage cells in the artery wall is believed to arise via the uptake and subsequent processing of modified low-density lipoproteins (LDL) via the endo-lysosomal system. In this study the effects of prolonged exposure to elevated glucose upon macrophage lysosomal function was examined to determine whether this contributes to modulated protein catabolism. Methods: Human monocytes were isolated from white-cell concentrates and differentiated, in vitro, into monocyte-derived macrophages over 11 days in medium containing 5-30 mmol/L glucose. Murine macrophage-like J774A.1 cells were incubated similarly. Lysosomal cathepsin (B, D, L and S) and acid lipase activities were assessed using fluorogenic substrates; cathepsin protein levels were examined by Western blotting. Lysosomal numbers were examined using the lysomotropic fluorescent dye LysoTracker DND-99, measurement of aryl sulfatase activity, and quantification of lysosome-associated membrane glycoprotein-1 (LAMP-1) by Western blotting. Results: Exposure to elevated glucose, but not mannitol, resulted in a concentration-dependent decrease in the activity, and to a lesser extent protein levels, of four lysosomal cathepsins. Acid lipase activity was also significantly reduced. Arysulfatase activity, LAMP-1 levels and lysosomal numbers were also decreased at the highest glucose concentrations, though to a lesser extent. Conclusion: Long term exposure of human and murine macrophage cells to elevated glucose levels result in a depression of lysosomal proteolytic and lipase activities. This may result in decreased clearance and cellular accumulation of (lipo)proteins and contribute to the accumulation of modified proteins and lipids in diabetes-associated atherosclerosis. © 2012 Elsevier Ireland Ltd

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma

FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7 alpha. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.Aldwin Suryo Rahmanto and Vasil Savov contributed equally to this work as first authorsAndrä Brunner, Sara Bolin and Holger Weishaupt contributed equally to this work as second authorsFredrik J Swartling and Olle Sangfelt contributed equally to this work as corresponding authors</p

CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer

CDK‐mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC‐driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer