Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Ubiquitylation of unphosphorylated c-myc by novel E3 ligase SCFFbxl8.

Overexpression of c-myc via increased transcription or decreased protein degradation is common to many cancer etiologies. c-myc protein degradation is mediated by ubiquitin-dependent degradation, and this ubiquitylation is regulated by several E3 ligases. The primary regulator is Fbxw7, which binds to a phospho-degron within c-myc. Here, we identify a new E3 ligase for c-myc, Fbxl8 (F-box and Leucine Rich Repeat Protein 8), as an adaptor component of the SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, for selective c-myc degradation. SCFFbxl8 binds and ubiquitylates c-myc, independent of phosphorylation, revealing that it regulates a pool of c-myc distinct from SCFFbxw7. Loss of Fbxl8 increases c-myc protein levels, protein stability, and cell division, while overexpression of Fbxl8 reduces c-myc protein levels. Concurrent loss of Fbxl8 and Fbxw7 triggers a robust increase in c-myc protein levels consistent with targeting distinct pools of c-myc. This work highlights new mechanisms regulating c-myc degradation.

Fbxl8 suppresses lymphoma growth and hematopoietic transformation through degradation of cyclin D3.

Overexpression of D-type cyclins in human cancer frequently occurs as a result of protein stabilization, emphasizing the importance of identification of the machinery that regulates their ubiqutin-dependent degradation. Cyclin D3 is overexpressed in ~50% of Burkitt's lymphoma correlating with a mutation of Thr-283. However, the E3 ligase that regulates phosphorylated cyclin D3 and whether a stabilized, phosphorylation deficient mutant of cyclin D3, has oncogenic activity are undefined. We describe the identification of SCF-Fbxl8 as the E3 ligase for Thr-283 phosphorylated cyclin D3. SCF-Fbxl8 poly-ubiquitylates p-Thr-283 cyclin D3 targeting it to the proteasome. Functional investigation demonstrates that Fbxl8 antagonizes cell cycle progression, hematopoietic cell proliferation, and oncogene-induced transformation through degradation of cyclin D3, which is abolished by expression of cyclin D3T283A, a non-phosphorylatable mutant. Clinically, the expression of cyclin D3 is inversely correlated with the expression of Fbxl8 in lymphomas from human patients implicating Fbxl8 functions as a tumor suppressor.

Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling.

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1ß expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

Consequences of interleukin 1ß-triggered chronic inflammation in the mouse prostate gland: Altered architecture associated with prolonged CD4+ infiltration mimics human proliferative inflammatory atrophy.

BACKGROUND: Elevated expression of the proinflammatory cytokine interleukin 1ß (IL-1ß) has been observed in expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome, and genetic polymorphisms associated with the IL1B gene are linked to increased risk for aggressive prostate cancer. METHODS: To study the role of IL-1ß expression in prostate inflammation, we examined IL1B expression in human prostatic proliferative inflammatory atrophy (PIA) lesions and developed a tetracycline-regulated human IL1B transgene in the mouse prostate. RESULTS: Here, we demonstrate that IL1B expression is a common finding in human PIA lesions, which harbored focal IL1B expression in epithelial and stromal compartments. Human IL1B expression in the mouse prostate elicited acute and chronic inflammation. Penetrance and expressivity were variable and tunable by altering transgene dosage and the presence of an exogenous inducible marker antigen (green fluorescent protein). Inflammation was characterized by infiltration of CD4+ T cells, demonstrating an adaptive immune response. Chronic inflammation persisted after doxycycline (Dox) withdrawal. Reactive epithelia increased expression of downstream cytokines, and altered glandular architecture was observed upon sustained induction of IL1B. Immunohistochemical analyses revealed a higher proliferative index and decreased Nkx3.1 expression in inflamed mouse prostates. CONCLUSIONS: These data implicate IL-1ß in human prostate pathology and this model provides a versatile platform to interrogate molecular mechanisms of inflammation-associated prostate pathologies associated with episodic or sustained IL-1ß expression.

Comprehensive urinary metabolomic characterization of a genetically induced mouse model of prostatic inflammation.

Dysfunction of the lower urinary tract commonly afflicts the middle-aged and aging male population. The etiology of lower urinary tract symptoms (LUTS) is multifactorial. Benign prostate hyperplasia, fibrosis, smooth muscle contractility, and inflammation likely contribute. Here we aim to characterize the urinary metabolomic profile associated with prostatic inflammation, which could inform future personalized diagnosis or treatment, as well as mechanistic research. Quantitative urinary metabolomics was conducted to examine molecular changes following induction of inflammation via conditional Interleukin-1ß expression in prostate epithelia using a novel transgenic mouse strain. To advance method development for urinary metabolomics, we also compared different urine normalization methods and found that normalizing urine samples based on osmolality prior to LC-MS most completely separated urinary metabolite profiles of mice with and without prostate inflammation via principal component analysis. Global metabolomics was combined with advanced machine learning feature selection and classification for data analysis. Key dysregulated metabolites and pathways were identified and were relevant to prostatic inflammation, some of which overlapped with our previous study of human LUTS patients. A binary classification model was established via the support vector machine algorithm to accurately differentiate control and inflammation groups, with an area-under-the-curve value of the receiver operating characteristic of 0.81, sensitivity of 0.974 and specificity of 0.995, respectively. This study generated molecular profiles of non-bacterial prostatic inflammation, which could assist future efforts to stratify LUTS patients and develop new therapies.