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Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.

Mistletoe (Viscum album) is a type of parasitic plant reported to have anticancer activity including in hepatocellular carcinoma (HCC). However, the mechanism of mistletoe's anticancer activity, and its effectiveness in treating HCC are not fully understood. We report here that mistletoe extracts, including Fraxini (grown on ash trees) and Iscador Q and M (grown on oak and maple trees), exert strong antiproliferative activity in Hep3B cells, with median inhibitory concentrations (IC 50 ) of 0.5 µg/mL, 7.49 µg/mL, and 7.51 µg/mL, respectively. Results of Reversed Phase Proteomic Array analysis (RPPA) suggests that Fraxini substantially down-regulates c-Myc expression in Hep3B cells. Fraxini-induced growth inhibition (at a concentration of 1.25 μg/ml) was less pronounced in c-Myc knockdown Hep3B cells than in control cells. Furthermore, in the Hep3B xenograft model, Fraxini-treated (8 mg/kg body weight) mice had significantly smaller tumors (34.6 ± 11.9 mm 3 ) than control mice (161.6 ± 79.4 mm 3 , p < 0.036). Similarly, c-Myc protein expression was reduced in Fraxini treated Hep3B cell xenografts compared to that of control mice. The reduction of c-Myc protein levels in vitro Hep3B cells appears to be mediated by the ubiquitin-proteasome system. Our results suggest the importance of c-Myc in Fraxini's antiproliferative activity, which warrants further investigation.

Despite the structural conservation of PTEN with dual-specificity phosphatases, there have been no reports regarding the regulatory mechanisms that underlie this potential dual-phosphatase activity. Here, we report that K27-linked polyubiquitination of PTEN at lysines 66 and 80 switches its phosphoinositide/protein tyrosine phosphatase activity to protein serine/threonine phosphatase activity. Mechanistically, high glucose, TGF-β, CTGF, SHH, and IL-6 induce the expression of a long non-coding RNA, GAEA (Glucose Aroused for EMT Activation), which associates with an RNA-binding E3 ligase, MEX3C, and enhances its enzymatic activity, leading to the K27-linked polyubiquitination of PTEN. The MEX3C-catalyzed PTEN K27-polyUb activates its protein serine/threonine phosphatase activity and inhibits its phosphatidylinositol/protein tyrosine phosphatase activity. With this altered enzymatic activity, PTEN K27-polyUb dephosphorylates the phosphoserine/threonine residues of TWIST1, SNAI1, and YAP1, leading to accumulation of these master regulators of EMT. Animals with genetic inhibition of PTEN K27-polyUb , by a single nucleotide mutation generated using CRISPR/Cas9 (Pten K80R/K80R ), exhibit inhibition of EMT markers during mammary gland morphogenesis in pregnancy/lactation and during cutaneous wound healing processes. Our findings illustrate an unexpected paradigm in which the lncRNA-dependent switch in PTEN protein serine/threonine phosphatase activity is important for physiological homeostasis and disease development.

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.