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SUMMARY The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adapter p62 contributes to mTORC1 activation in response to amino acids, and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63-polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation.

SUMMARY Intestinal epithelial homeostasis requires continuous renewal supported by stem cells located in the base of the crypt. Disruption of this balance results in failure to regenerate and initiates tumorigenesis. The β-catenin and Yap pathways in Lgr5+ stem cells have been shown to be central to this process. However, the precise mechanisms by which these signaling molecules are regulated in the stem cell population are not totally understood. PKCζ has been previously demonstrated to be a negative regulator of intestinal tumorigenesis. We show here that PKCζ suppresses intestinal stem cell function by promoting the downregulation of β-catenin and Yap through direct phosphorylation. PKCζ deficiency results in increased stem cell activity in organoid cultures and in vivo, accounting for the increased tumorigenic and regenerative activity response of Lgr5+-specific PKCζ-deficient mice. This demonstrates that PKCζ is central to the control of stem cells in intestinal cancer and homeostasis.

Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches–such as selected reaction monitoring (SRM)–as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.