PDF file - 500KB, Supplemental Figure 1. Upregulation of hMOB3 in GBM and characterization of hMOB3 antibody and shRNAs.

Supplementary Table 1 from MAP Kinase-Interacting Kinase 1 Regulates SMAD2-Dependent TGF-β Signaling Pathway in Human Glioblastoma

PDF file - 323KB, Supplemental Figure 2. hMOB3 specifically interacts with MST1.

Supplementary Figure 2 from Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Supplementary Figure Legends 1-7, Methods from Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Supplementary Figures 1-4 from MAP Kinase-Interacting Kinase 1 Regulates SMAD2-Dependent TGF-β Signaling Pathway in Human Glioblastoma

PDF file - 280KB, Supplemental Figure 3. hMOB3 negatively regulates MST1 apoptotic cleavage.

Supplementary Figure 7 from Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Background:Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results.Methods:Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment.Results:SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration.Conclusions:Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.

Phosphorylation is a post-translational protein modification regulating most known cellular processes. While protein kinases constitute a large family of highly conserved enzymes, identification of active kinases is challenging due to a low abundance of some of these signaling molecules. Although chicken is the first agricultural animal to have a sequenced genome, annotation of the kinome, i.e., a complement of all protein kinases in the genome is limited. We used chemical probes consisting of ATP and ADP derivatives binding to specific lysine (Lys) residues within the ATP-binding pocket of kinases, combined with proteomics, to identify 267 peptides labeled with the ATP and ADP acyl derivatives and 188 corresponding chicken kinases in chicken spleen and liver. Our description of active chicken kinases and ATP binding sites will support future studies focused on identifying the role of this important class of enzymes in chicken health and disease.Advances made in understanding chicken enzymes are critical for the improved knowledge of the regulatory pathways controlling physiological processes in chicken. Since protein phosphorylation controls multiple aspects of cell fate, it is often linked to pathological conditions, and understanding of the kinase expression in chicken is essential for future therapeutic approaches. We coupled proteomics and labeling with active-site probes binding to Lys residues within the ATP-binding pocket of kinases to identify 188 kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. Results of the present study describing catalytically active kinases is a starting point for chemoproteomic-based interrogation of kinases in chicken exposed to different conditions. Kinases identified in this study are available through the Chickspress genome browser that has previously published mRNA, miRNA, and shotgun proteomics data.

Chronic inflammation is a major cause of human cancer. Clinical cancer therapies against inflammatory risk factors are strategically determined. To rationally guide a novel drug development, an improved mechanistic understanding on the pathological connection between inflammation and carcinogenesis is essential. PI3K-PKB signaling axis has been extensively studied and shown to be one of the key oncogenic drivers in most types of cancer. Pharmacological inhibition of the components along this signaling axis is of great interest for developing novel therapies. Interestingly, emerging studies have shown a close association between PKB activation and inflammatory activity in the vicinity of the tumor, and either blockade of PKB or attenuation of para-tumoral inflammation reveals a mutual-interactive pattern through pathway crosstalk. In this review, we intend to discuss recent advances of PKB-regulated chronic inflammation and its potential impacts on tumor development.

BRAF inhibitors (BRAFi) and the combination therapy of BRAF and MEK inhibitors (MEKi) were recently approved for therapy of metastatic melanomas harbouring the oncogenic BRAFV600 mutation. Although these therapies have shown pronounced therapeutic efficacy, the limited durability of the response indicates an acquired drug resistance that still remains mechanistically poorly understood at the molecular level. We conducted transcriptome gene profiling in BRAFi-treated melanoma cells and identified that Mer tyrosine kinase (MerTK) is specifically upregulated. MerTK overexpression was demonstrated not only in melanomas resistant to BRAFi monotherapy (5 out of 10 samples from melanoma patients) but also in melanoma resistant to BRAFi+MEKi (1 out of 3), although MEKi alone does not affect MerTK. Mechanistically, BRAFi-induced activation of Zeb2 stimulates MerTK in BRAFV600 melanoma through mTORC1-triggered activation of autophagy. Co-targeting MerTK and BRAFV600 significantly reduced tumour burden in xenografted mice, which was pheno-copied by co-inhibition of autophagy and mutant BRAFV600.

Unopposed phosphoinositide 3-kinase (PI3K) activity and 3-phosphoinositide production in Jurkat cells, due to a mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor-suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB. In Jurkat cells, PKB is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3-Phosphoinositide-dependent protein kinase-1 (PDK1), an enzyme that also contains a PH domain, catalyses Thr308 phosphorylation of PKB in addition to other kinase families such as PKC isoforms. It is unknown, however, whether the loss of PTEN in Jurkat cells also results in unregulated PDK1 activity and whether such loss has an impact on activation-loop phosphorylation of other PDK1 substrates e.g. PKC. In this study, we addressed whether loss of PTEN in Jurkat cells affects PDK1 catalytic activity and intracellular localization. We demonstrated that reducing the level of 3-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3K or expression of PTEN does not affect PDK1 activity or its intracellular localization. We conclude, therefore, that although Jurkat cells lack PTEN expression, only a subset of pathways downstream of PDK1 are perturbed as a consequence of PTEN loss.

Unopposed phosphoinositide 3-kinase (PI3K) activity and 3-phosphoinositide production in Jurkat cells, due to a mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor-suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB. In Jurkat cells, PKB is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3-Phosphoinositide-dependent protein kinase-1 (PDK1), an enzyme that also contains a PH domain, catalyses Thr308 phosphorylation of PKB in addition to other kinase families such as PKC isoforms. It is unknown, however, whether the loss of PTEN in Jurkat cells also results in unregulated PDK1 activity and whether such loss has an impact on activation-loop phosphorylation of other PDK1 substrates e.g. PKC. In this study, we addressed whether loss of PTEN in Jurkat cells affects PDK1 catalytic activity and intracellular localization. We demonstrated that reducing the level of 3-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3K or expression of PTEN does not affect PDK1 activity or its intracellular localization. We conclude, therefore, that although Jurkat cells lack PTEN expression, only a subset of pathways downstream of PDK1 are perturbed as a consequence of PTEN loss.

Although modern therapeutic strategies have brought significant progress to cancer care in the last 30 years, drug resistance to targeted monotherapies has emerged as a major challenge. Aberrant regulation of multiple physiological signaling pathways indispensable for developmental and metabolic homeostasis, such as hyperactivation of pro-survival signaling axes, loss of suppressive regulations, and impaired functionalities of the immune system, have been extensively investigated aiming to understand the diversity of molecular mechanisms that underlie cancer development and progression. In this review, we intend to discuss the molecular mechanisms of how conventional physiological signal transduction confers to acquired drug resistance in cancer patients. We will particularly focus on protooncogenic receptor kinase inhibition-elicited tumor cell adaptation through two major core downstream signaling cascades, the PI3K/Akt and MAPK pathways. These pathways are crucial for cell growth and differentiation and are frequently hyperactivated during tumorigenesis. In addition, we also emphasize the emerging roles of the deregulated host immune system that may actively promote cancer progression and attenuate immunosurveillance in cancer therapies. Understanding these mechanisms may help to develop more effective therapeutic strategies that are able to keep the tumor in check and even possibly turn cancer into a chronic disease.

The transcription factor Twist is an important regulator of cranial suture during embryogenesis. Closure of the neural tube is achieved via Twist-triggered cellular transition from an epithelial to mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT), characterized by a remarkable increase in cell motility. In the absence of Twist activity, EMT and associated phenotypic changes in cell morphology and motility can also be induced, albeit moderately, by other transcription factor families, including Snail and Zeb. Aberrant EMT triggered by Twist in human mammary tumour cells was first reported to drive metastasis to the lung in a metastatic breast cancer model. Subsequent analysis of many types of carcinoma demonstrated overexpression of these unique EMT transcription factors, which statistically correlated with worse outcome, indicating their potential as biomarkers in the clinic. However, the mechanisms underlying their activation remain unclear. Interestingly, increasing evidence indicates they are selectively activated by distinct intracellular kinases, thereby acting as downstream effectors facilitating transduction of cytoplasmic signals into nucleus and reprogramming EMT and mesenchymal-epithelial transition (MET) transcription to control cell plasticity. Understanding these relationships and emerging data indicating differential phosphorylation of Twist leads to complex and even paradoxical functionalities, will be vital to unlocking their potential in clinical settings.

Current standard-of-care treatment for malignant cancers includes radiotherapy and adjuvant chemotherapy. Here, we report increased MAP kinase-interacting kinase (MNK)-regulated phosphorylation of translation initiation factor 4E (eIF4E) in glioma cells upon temozolomide (TMZ) treatment and in medullary thyroid carcinoma (MTC) cells in response to targeted radionuclide therapy. Depletion of MNK activity by using two MNK inhibitors, CGP57380 or cercosporamide, as well as by MNK1-specific knockdown sensitized glioblastoma (GBM) cells and GBM-derived spheres to TMZ. Furthermore, CGP57380 treatment enhanced response of MTC cells to (177)Lu-labeled gastrin analogue. In order to understand how MNK signaling pathways support glioma survival we analyzed putative MNK substrates by quantitative phosphoproteomics in normal condition and in the presence of TMZ. We identified MNK inhibitor-sensitive phosphorylation sites on eIF4G1, mutations of which either influenced eIF4E phosphorylation or glioma cell response to TMZ, pointing to altered regulation of translation initiation as a resistance mechanism. Pharmacological inhibition of overexpressed MNK1 by CGP57380 reduced eIF4E phosphorylation and induced association of inactive MNK1 with eIF4G1. Taken together, our data show an activation of MNK-mediated survival mechanisms in response to either glioma chemotherapy or MTC targeted radiation and suggest that inhibition of MNK activity represents an attractive sensitizing strategy for cancer treatments.

Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.

BACKGROUND:Phosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown.RESULTS:We show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation of NDR1/2 from the intestinal epithelium renders mice exquisitely sensitive to chemically induced colon carcinogenesis. Analysis of human colon cancer samples further revealed that NDR2 and YAP1 protein expression are inversely correlated in the majority of samples with high YAP1 expression. Collectively, we report NDR1/2 as physiological YAP1-S127 kinases that might function as tumor suppressors upstream of YAP1 in human colorectal cancer.CONCLUSIONS:We establish mammalian NDR1/2 as bona fide kinases that target YAP1 on S127 in vitro and in vivo. Our findings therefore have important implications for a broad range of research efforts aimed at decoding and eventually manipulating YAP1 biology in cancer settings, regenerative medicine, and possibly also noncancer human diseases.