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

The role of hepatocyte growth factor/scatter factor in hepatoblastoma and hepatocellular carcinoma progression

Hepatocyte growth factor/scatter factor (HGF/SF) is a ubiquitously expressed molecule that elicits pleiotropic functions on epithelial cells, including mitogenic, motogenic, differentiating, angiogenic, and morphogenic effects. The receptor for HGF/SF is c-Met, a product of the proto-oncogene c-met, which is abundantly expressed in many tumors, rendering them highly receptive for HGF/SF signals. In hepatoblastoma (HB) and hepatocellular carcinoma (HCC), a high relapse incidence and post-operative residual tumor growth can be detected, when the serum levels of HGF/SF are markedly elevated, suggesting a link between this molecule and tumor malignancy. Although HB and HCC are two distinct subtypes of primary tumors arising from liver parenchymal cells and thus differ by many histo-clinical characteristics, comparative analysis of the impact of HGF/SF on these tumors may provide some clues on the oncogenic pathways leading to liver tumor progression. The results of this study demonstrate that HGF/SF mediates cytoprotective functions against the apoptotic inducers cisplatin, camptothecin, and starvation in a phosphoinositide 3-kinase (PI3K)dependent manner, thereby contributing to chemotherapy resistance. Differences between HB and HCC cells regarding the sensitivity towards HGF/SF and HGF/SFstimulated cellular responses were observed and are associated with c-Met expression. Furthermore, our experiments demonstrate that HGF/SF is a potent inducer of cell scattering and migration. HGF/SF triggers scattering of epithelial cells by upregulating the expression of Snail, a transcriptional repressor involved in epithelial-mesenchymal transition (EMT). Snail, which represses for example the expression of E-cadherin and claudin-3, is required for HGF/SF-induced cell scattering, since shRNA-mediated ablation of Snail expression prevents this process. HGF/SF-induced upregulation of Snail transcription involves activation of the mitogen-activated protein kinase (MAPK) pathway and requires the activity of early growth response factor (Egr)-1. Thus, HGF/SF plays a critical role in cell scattering, migration, and invasion. Together, these findings highlight the importance of HGF/SF in tumor cell survival and tumor progression and suggest that it should be considered as a candidate for therapeutic strategies

Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets

Hepatocyte growth factor induces cell scattering through MAPK/Egr-1-mediated upregulation of Snail

Hepatocyte growth factor/scatter factor (HGF) exerts several functions in physiological and pathological processes, among them the induction of epithelial cell scattering and motility. Its pivotal role in angiogenesis, tumor progression, and metastasis is evident; however, the underlying molecular mechanisms are still poorly understood. Here, we demonstrate that HGF induces scattering of epithelial cells by upregulating the expression of Snail, a transcriptional repressor involved in epithelial–mesenchymal transition (EMT). Snail is required for HGF-induced cell scattering, since shRNA-mediated ablation of Snail expression prevents this process. HGF-induced upregulation of Snail transcription involves activation of the mitogen-activated protein kinase (MAPK) pathway and requires the activity of early growth response factor-1 (Egr-1). Upon induction by Egr-1, Snail represses the expression of E-cadherin and claudin-3 genes. It also binds to the Egr-1 promoter and represses Egr-1 transcription, thereby establishing a negative regulatory feedback loop. These findings indicate that Snail upregulation by HGF is mediated via the MAPK/Egr-1 signaling pathway and that both Snail and Egr-1 play a critical role in HGF-induced cell scattering, migration, and invasion

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