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Summary β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity., Graphical Abstract, Highlights • Gain-of-function kinome screen identifies AAK1 as a repressor of WNT signaling • AAK1 promotes clathrin-mediated endocytosis of LRP6 • Selective AAK1 inhibitor stabilizes β-catenin and activates WNT signaling • WNT induces AAK1-dependent phosphorylation of AP2M1 and LRP6 endocytosis, WNT signal transduction is essential for normal development and contributes to many human diseases. Agajanian et al. used a kinase gain-of-function screen to show that WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of the WNT receptor. This work identifies an AAK-driven negative feedback loop that downregulates WNT signaling.

Most aspects of leukocyte physiology are under the control of reversible tyrosine phosphorylation. It is clear that excessive phosphorylation of signal transduction elements is a pivotal element of many different pathologies including haematological malignancies and accordingly, strategies that target such phosphorylation have clinically been proven highly successful for treatment of multiple types of leukemias and lymphomas. Cellular phosphorylation status is dependent on the resultant activity of kinases and phosphatases. The cell biology of the former is now well understood: for most cellular phosphoproteins we now know the kinases responsible for their phosphorylation and we understand the principles of their aberrant activity in disease. With respect to phosphatases, however, our knowledge is much patchier. Although the sequences of whole genomes allow us to identify phosphatases using in silico methodology, whereas transcription profiling allows us to understand how phosphatase expression is regulated during disease, most functional questions as to substrate specificity, dynamic regulation of phosphatase activity and potential for therapeutic intervention are still to a large degree open. Nevertheless, recent studies have allowed us to make meaningful statements on the role of tyrosine phosphatase activity in the three major signaling pathways that are commonly affected in leukemias, i.e. the Ras-Raf-ERK1/2, the Jak-STAT and the PI3K-PKB-mTOR pathways. Lessons learned from these pathways may well be applicable elsewhere in leukocyte biology as well. (C) 2010 Elsevier B.V. All rights reserved.

Background: Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. Objective: To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. Design, setting, and participants: Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. Outcome measurements and statistical analysis: The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. Results and limitations: LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]: 1.99; p < 0.001) and reduced patient survival (HR: 1.53; p = 0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. Conclusions: Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. Patient summary: These findings implicate low-molecular-weight protein tyrosine phosphatase as a novel oncogene in prostate cancer and could offer the possibility of using this protein as biomarker or target for treatment of this disease. (C) 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-beta production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-beta expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.

Pancreatic cancer ranks fourth among cancer-related causes of death in North America. Minimal progress has been made in the diagnosis and treatment of patients with late-stage tumors. Moreover, pancreatic cancer aggressiveness is closely related to high levels of pro-survival mediators, which can ultimately lead to rapid disease progression, resistance and metastasis. The main goal of this study was to define the mechanisms by which calix[6]arene, but not other calixarenes, efficiently decreases the aggressiveness of a drug resistant human pancreas carcinoma cell line (Panc-1). Calix[6]arene was more potent in reducing Panc-1 cell viability than gemcitabine and 5-fluorouracil. In relation to the underlying mechanisms of cytotoxic effects, it led to cell cycle arrest in the G0/G1 phase through downregulation of PIM1, CDK2, CDK4 and retinoblastoma proteins. Importantly, calix[6]arene abolished signal transduction of Mer and AXL tyrosine kinase receptors, both of which are usually overexpressed in pancreatic cancer. Accordingly, inhibition of PI3K and mTOR was also observed, and these proteins are positively modulated by Mer and AXL. Despite decreasing the phosphorylation of AKT at Thr308, calix[6]arene caused an increase in phosphorylation at Ser473. These findings in conjunction with increased BiP and IRE1-α provide a molecular basis explaining the capacity of calix[6]arene to trigger endoplasmic reticulum stress and autophagic cell death. Our findings highlight calix[6]arene as a potential candidate for overcoming pancreatic cancer aggressiveness. Importantly, we provide evidence that calix[6]arene affects a broad array of key targets that are usually dysfunctional in pancreatic cancer, a highly desirable characteristic for chemotherapeutics.

The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML.

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+)/c-Kit(+)/P-glycoprotein(+)/MRP1(+) TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.

Item does not contain fulltext Mounting evidence suggests an important role for CCAAT-enhancer binding protein delta (C/EBPdelta) in the acute-phase response after bacterial infection. However, whether C/EBPdelta limits pneumonia remains elusive and is the aim of this study. Therefore, bacterial outgrowth, inflammatory responses, inflammatory cell influx, and survival were assessed in wild-type and C/EBPdelta(-/-) mice infected with Klebsiella pneumoniae via the airways. We showed that C/EBPdelta expression is highly induced in the lung during pulmonary infection and that Klebsiella-induced mortality was significantly increased among C/EBPdelta(-/-) mice. Bacterial loads and inflammatory responses were similar in wild-type and C/EBPdelta(-/-) mice early during infection, whereas bacterial loads were increased in C/EBPdelta(-/-) mice later during infection. Moreover, macrophage numbers were reduced in lungs of C/EBPdelta(-/-) mice. In vitro experiments showed that C/EBPdelta only slightly affects macrophage function. Our data thus show that C/EBPdelta contributes to host defense against Klebsiella-induced pneumonia and suggests that C/EBPdelta-dependent macrophage mobilization is a key mechanism.

Natural-food-based compounds show substantial promise for prevention and biotherapy of cancers including leukemia. In general, their mechanism of action remains unclear, hampering rational use of these compounds. Herein we show that the common dietary flavonoid apigenin has anticancer activity, but also may decrease chemotherapy sensitivity, depending on the cell type. We analyzed the molecular consequences of apigenin treatment in two types of leukemia, the myeloid and erythroid subtypes. Apigenin blocked proliferation in both lineages through cell-cycle arrest in G(2)/M phase for myeloid HL60 and G(0)/G(1) phase for erythroid TF1 cells. In both cell lines the JAK/STAT pathway was one of major targets of apigenin. Apigenin inhibited PI3K/PKB pathway in HL60 and induced caspase-dependent apoptosis. In contrast, no apoptosis was detected in TF1 cells, but initiation of autophagy was observed. The block in cell cycle and induction of autophagy observed in this erythroleukemia cell line resulted in a reduced susceptibility toward the commonly used therapeutic agent vincristine. Thus, this study shows that although apigenin is a potential chemopreventive agent due to the induction of leukemia cell-cycle arrest, caution in dietary intake of apigenin should be taken during disease as it potentially interferes with cancer treatment. Cell Death and Disease (2010) 1, e19; doi:10.1038/cddis.2009.18; published online 28 January 2010