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The colony-stimulating factor-1 (CSF-1) and its receptor CSF-1R physiologically regulate the monocyte/macrophage system, trophoblast implantation and breast development. An abnormal CSF-1R expression has been documented in several human epithelial tumors, including breast carcinomas. We recently demonstrated that CSF-1/CSF-1R signaling drives proliferation of breast cancer cells via 'classical' receptor tyrosine kinase signaling, including activation of the extracellular signal-regulated kinase 1/2. In this paper, we show that CSF-1R can also localize within the nucleus of breast cancer cells, either cell lines or tissue specimens, irrespectively of their intrinsic molecular subtype. We found that the majority of nuclear CSF-1R is located in the chromatin-bound subcellular compartment. Chromatin immunoprecipitation revealed that CSF-1R, once in the nucleus, binds to the promoters of the proliferation-related genes CCND1, c-JUN and c-MYC. CSF-1R also binds the promoter of its ligand CSF-1 and positively regulates CSF-1 expression. The existence of such a receptor/ligand regulatory loop is a novel aspect of CSF-1R signaling. Moreover, our results provided the first evidence of a novel localization site of CSF-1R in breast cancer cells, suggesting that CSF-1R could act as a transcriptional regulator on proliferation-related genes. [ABSTRACT FROM AUTHOR]

Growth hormone (GH) has long been known to be the body's primary regulator of body growth and a regulator of metabolism, yet the mechanisms by which GH regulates the transcription of specific genes required for these processes are just now being delineated. GH binding to its receptor recruits and activates the receptor-associated JAK2 that in turn phosphorylates tyrosines within itself and the GH receptor. These tyrosines form binding sites for a number of signaling proteins, including members of the family of signal transducers and activators of transcription (STAT). Among the known signaling molecules for GH, STAT proteins play a particularly prominent role in the regulation of gene transcription. This paper will review what is currently understood about which STAT proteins are regulated by GH, how they are regulated by GH, the GH-dependent genes they regulate, and discuss current theories about how GH-activated STAT signaling is regulated. Particular attention will be given to the novel role that STAT5 plays in sexually dimorphic gene expression in the liver as determined by the secretory pattern of GH and the role of STAT5 in body growth. Oncogene (2000) 19, 2585–2597 [ABSTRACT FROM AUTHOR]

Communication between cells is often mediated by secreted signaling molecules that bind cell surface receptors and modulate the activity of specific intracellular effectors. The Wnt family of secreted glycoproteins is one group of signaling molecules that has been shown to control a variety of developmental processes including cell fate specification, cell proliferation, cell polarity and cell migration. In addition, mis-regulation of Wnt signaling can cause developmental defects and is implicated in the genesis of several human cancers. The importance of Wnt signaling in development and in clinical pathologies is underscored by the large number of primary research papers examining various aspects of Wnt signaling that have been published in the past several years. In this review, we will present a synopsis of current research with particular attention paid to molecular mechanism of Wnt signal transduction and how the mis-regulation of Wnt signaling leads to cancer. [ABSTRACT FROM AUTHOR]

This article serves as an introduction to the collection of reviews on nuclear factor-kappaB (NF-κB). It provides an overview of the discovery and current status of NF-κB as a research topic. Described are the structures, activities and regulation of the proteins in the NF-κB family of transcription factors. NF-κB signaling is primarily regulated by inhibitor κB (IκB) proteins and the IκB kinase complex through two major pathways: the canonical and non-canonical NF-κB pathways. The organization and focus of articles included in the following reviews are described, as well as likely future areas of research interest on NF-κB.Oncogene (2006) 25, 6680–6684. doi:10.1038/sj.onc.1209954 [ABSTRACT FROM AUTHOR]

Necroptosis represents a key programmed cell death pathway involved in various physiological and pathophysiological conditions. However, the role of reactive oxygen species (ROS) in necroptotic signaling has remained unclear. In the present study, we identify ROS as critical regulators of BV6/tumor necrosis factor-α (TNFα)-induced necroptotic signaling and cell death. We show that BV6/TNFα-induced cell death depends on ROS production, as several ROS scavengers such as butylated hydroxyanisole, N-acetylcysteine, α-tocopherol and ethyl pyruvate significantly rescue cell death. Before cell death, BV6/TNFα-stimulated ROS generation promotes stabilization of the receptor-interacting protein kinase 1 (RIP1)/RIP3 necrosome complex via a potential positive feedback loop, as on the one hand radical scavengers attenuate RIP1/RIP3 necrosome assembly and phosphorylation of mixed lineage kinase domain like (MLKL), but on the other hand silencing of RIP1 or RIP3 reduces ROS production. Although MLKL knockdown effectively decreases BV6/TNFα-induced cell death, it does not affect RIP1/RIP3 interaction and only partly reduces ROS generation. Moreover, the deubiquitinase cylindromatosis (CYLD) promotes BV6/TNFα-induced ROS generation and necrosome assembly even in the presence of BV6, as CYLD silencing attenuates these events. Genetic silencing of phosphoglycerate mutase 5 or dynamin-related protein 1 (Drp1) fails to protect against BV6/TNFα-induced cell death. By demonstrating that ROS are involved in regulating BV6/TNFα-induced necroptotic signaling, our study provides new insights into redox regulation of necroptosis. [ABSTRACT FROM AUTHOR]

Specific protein-protein interactions (PPIs) constitute a key underlying mechanism for the presence of a multitude of intracellular signaling pathways, which are essential for the survival of normal and cancer cells. Specific molecular blockers for a crucial PPI would therefore be invaluable tools for an enhanced functional interrogation of the signaling pathway harboring this particular PPI. On the other hand, if a particular PPI is essential for the survival of cancer cells but is absent in or dispensable for the survival of normal cells, its specific molecular blockers could potentially be developed into effective anticancer therapeutics. Due to the flat and extended PPI interface, it would be conceivably difficult for small molecules to achieve an effective blockade, a problem which could be potentially circumvented with peptides or proteins. However, the well-documented proteolytic instability and cellular impermeability of peptides and proteins in general would make their developing into effective intracellular PPI blockers quite a challenge. With the advent of the peptide 'stapling' technology which was demonstrated to be able to stabilize the α-helical conformation of a peptide via bridging two neighboring amino-acid side chains with a 'molecular staple', a linear parent peptide could be transformed into a stronger PPI blocker with enhanced proteolytic stability and cellular permeability. This review will furnish an account on the peptide 'stapling' technology and its exploitation in efforts to achieve an enhanced functional interrogation or manipulation of intracellular signaling pathways especially those that are cancer relevant. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is a cellular sensor that controls cytosolic DNA-activated innate immune signaling. We have previously demonstrated that STING-deficient mice are resistant to carcinogen-induced skin cancer, similar to myeloid differentiation primary response gene 88 (MyD88) deficient mice, since the production of STING-dependent DNA-damage-induced proinflammatory cytokines, that likely require MyD88 signaling to exert their growth-promoting activity, are prevented. In contrast, MyD88-deficient mice are sensitive to colitis-associated cancer (CAC), since selected cytokines generated following DNA-damage also activate repair pathways, which can help prevent tumor development. Here, we demonstrate that STING signaling facilitates wound repair processes and that analogous to MyD88-deficient mice, STING-deficient mice (SKO) are prone to CAC induced by DNA-damaging agents. SKO mice harboring tumors exhibited low levels of tumor-suppressive interleukin-22 binding protein (IL-22BP) compared to normal mice, a cytokine considered critical for preventing colon-related cancer. Our data indicate that STING constitutes a critical component of the host early response to intestinal damage and is essential for invigorating tissue repair pathways that may help prevent tumorigenesis. [ABSTRACT FROM AUTHOR]

Tumour cells often acquire the ability to escape cell death, a key event leading to the development of cancer. In almost half of all human cancers, the capability to induce cell death is reduced by the mutation and inactivation of p53, a tumour suppressor protein that is a central regulator of apoptosis. As a result, there is a crucial need to identify different cell death pathways that could be targeted in malignancies lacking p53. p73, the closely related p53 family member, can regulate many p53 target genes and therefore some of the same cellular responses as p53. Unlike p53, however, p73 is seldom mutated in cancer, making it an attractive, alternative death effector to target. We report here the ability of p73 to upregulate the expression of the A2B receptor, a recently characterized p53 target that effectively promotes cell death in response to extracellular adenosine-a metabolite that accumulates during various forms of cellular stress. Importantly, we show that p73-dependent stimulation of A2B signalling markedly enhances apoptosis in cancer cells that are devoid of p53. This mode of death is caspase- and puma-dependent, and can be prevented by the overexpression of anti-apoptotic Bcl-XL. Moreover, treatment of p53-null cancer cells with the chemotherapeutic drug adriamycin (doxorubicin) induces A2B in a p73-dependent manner and, in combination with an A2B agonist, substantially enhances apoptotic death. We therefore propose an alternate and distinct p53-independent pathway to stimulate programmed cell death involving p73-mediated engagement of adenosine signalling. [ABSTRACT FROM AUTHOR]

Multiform glioblastomas (GBM) are the most frequent and aggressive primary brain tumors in adults. The poor prognosis is due to neo-angiogenesis and cellular invasion, processes that require complex chemotaxic mechanisms involving motility, migration and adhesion. Understanding these different cellular events implies identifying receptors and transduction pathways that lead to and promote either migration or adhesion. Here we establish that glioma express the vasoactive peptide urotensin II (UII) and its receptor UT and that UT-mediated signaling cascades are involved in glioma cell migration and adhesion. Components of the urotensinergic systems, UII and UT, are widely expressed in patient-derived GBM tissue sections, glioma cell lines and fresh biopsy explants. Interestingly, gradient concentrations of UII produced chemoattracting migratory/motility effects in glioma as well as HEK293 cells expressing human UT. These effects mainly involved the G13/Rho/rho kinase pathway while partially requiring Gi/o/PI3K components. In contrast, we observed that homogeneous concentrations of UII drastically blocked cell motility and stimulated cell-matrix adhesions through a UT/Gi/o signaling cascade, partially involving phosphatidylinositol-3 kinase. Finally, we provide evidence that, in glioma cells, homogeneous concentration of UII allowed translocation of Gα13 to the UT receptor at the plasma membrane and increased actin stress fibers, lamellipodia formation and vinculin-stained focal adhesions. UII also provoked a re-localization of UT precoupled to Gαi in filipodia and initiated integrin-stained focal points. Altogether, these findings suggest that UT behaves as a chemotaxic receptor, relaying a signaling switch between directional migration and cell adhesion under gradient or homogeneous concentrations, thereby redefining sequential mechanisms affecting tumor cells during glioma invasion. Taken together, our results allow us to propose a model in order to improve the design of compounds that demonstrate signaling bias for therapies that target specifically the Gi/o signaling pathway. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor that has a high propensity for metastases. Through osteoblast-specific alteration of p53 status, we developed a genetically engineered mouse model of localized and metastatic OS to gain an understanding into the molecular pathogenesis of OS. Microarray analysis of both localized tumors and metastatic tumors identified the downregulation of the naked cuticle homolog 2 (NKD2) gene, a negative regulator of Wnt signaling. Overexpression of NKD2 in metastatic human and mouse OS cells significantly decreases cell proliferation, migration and invasion ability in vitro and drastically diminishes OS tumor growth and metastasis in vivo, whereas downregulation enhances migratory and invasive potential. Evaluation of NKD2-overexpressing tumors revealed upregulation of tumor-suppressor genes and downregulation of molecules involved in blood vessel formation and cell migration. Furthermore, assessment of primary human OS revealed downregulation of NKD2 in metastatic and recurrent OS. Finally, we provide biological evidence that use of small-molecule inhibitors targeting the Wnt pathway can have therapeutic efficacy in decreasing metastatic properties in OS. Our studies provide compelling evidence that downregulation of NKD2 expression and alterations in associated regulated pathways have a significant role in driving OS tumor growth and metastasis. [ABSTRACT FROM AUTHOR]

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in β-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced β-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting β-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/β-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/β-catenin signalings during oncogenesis. [ABSTRACT FROM AUTHOR]

The four R-spondins (RSPO1-4) and their three related receptors LGR4, 5 and 6 (LGR4-6) have emerged as a major ligand-receptor system with critical roles in development and stem cell survival through modulation of Wnt signaling. Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of human colorectal cancer. However, the exact roles and mechanisms of the RSPO-LGR system in oncogenesis remain largely unknown. We found that RSPO3 is aberrantly expressed at high levels in approximately half of Keap1-mutated lung adenocarcinomas (ADs). This high RSPO3 expression is driven by a combination of demethylation of its own promoter region and deficiency in Keap1 instead of gene fusion as in colon cancer. Patients with RSPO3-high tumors (~9%, 36/412) displayed much poorer survival than the rest of the cohort (median survival of 28 vs 163 months, log-rank test P<0.0001). Knockdown (KD) of RSPO3, LGR4 or their signaling mediator IQGAP1 in lung cancer cell lines with Keap1 deficiency and high RSPO3-LGR4 expression led to reduction in cell proliferation and migration in vitro, and KD of LGR4 or IQGAP1 resulted in decrease in tumor growth and metastasis in vivo. These findings suggest that aberrant RSPO3-LGR4 signaling potentially acts as a driving mechanism in the aggressiveness of Keap1-deficient lung ADs. [ABSTRACT FROM AUTHOR]

Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm2) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels. In the present study, we demonstrate that Syk interacts with the integrin β4 subunit (ITGβ4) of integrin α6β4 in epithelial cells and that concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. We find that both Syk and LMP2A have an effect on ITGβ4 cell surface expression. However, in LMP2A expressing cells, ITGβ4 remains concentrated at the cellular protrusions, an expression pattern characteristic of motile cells, including NPC-derived epithelial cells. This effect of LMP2A on ITGβ4 localization is associated with a greater propensity for migration and invasion in-vitro, and may contribute to the invasive property of LMP2A-expressing NPC. [ABSTRACT FROM AUTHOR]

The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: <10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb repressive complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models, and co-ordinated upregulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2-regulated genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonization. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2-dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis. [ABSTRACT FROM AUTHOR]

Aberrant activation of Notch signaling has an essential role in colorectal cancer (CRC) progression. Amplified in breast cancer 1 (AIB1), also known as steroid receptor coactivator 3 or NCOA3, is a transcriptional coactivator that promotes cancer cell proliferation and invasiveness. However, AIB1 implication in CRC progression through enhancing Notch signaling is unknown. In this study, we found that several CRC cell lines expressed high levels of AIB1, and knockdown of AIB1 decreased cell proliferation, colony formation and tumorigenesis of these CRC cells. Specifically, knockdown of AIB1 inhibited cell cycle progression at G1 phase by decreasing the mRNA levels of cyclin A2, cyclin B1, cyclin E2 and hairy and enhancer of split (Hes) 1. Furthermore, AIB1 interacted with Notch intracellular domain and Mastermind-like 1 and was recruited to the Hes1 promoter to enhance Notch signaling. Downregulation of AIB1 also decreased CRC cell invasiveness in vitro and lung metastasis in vivo. Besides that, knockout of AIB1 in mice inhibited colon carcinogenesis induced by azoxymethane/dextran sodium sulfate treatment. The mRNA levels of cyclin B1 and Hes5 were downregulated, but p27, ATOH1 and MUC2 were upregulated in the colon tumors from AIB1-deficient mice compared with those from wild-type mice. Thus, our results signify the importance of AIB1 in CRC and demonstrate that AIB1 promotes CRC progression at least in part through enhancing Notch signaling, suggesting that AIB1 is a potential molecular target for CRC treatment. [ABSTRACT FROM AUTHOR]

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage. [ABSTRACT FROM AUTHOR]

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor-infiltrating myeloid cells (TIMs) are known to promote metastasis, but the mechanisms underlying their collaboration with tumor cells remain unknown. Here, we report an oncogenic role for microRNA (miR) in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss-of-function approach utilizing selective depletion of the miR-processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the downregulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3, whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer, and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling. [ABSTRACT FROM AUTHOR]

Androgen receptor splicing variants (ARVs) that lack the ligand-binding domain (LBD) are associated with the development of castration-resistant prostate cancer (CRPC), including resistance to the new generation of high-affinity anti-androgens. However, the mechanism by which ARV expression is regulated is not fully understood. In this study, we show that the activation of classical nuclear factor-kappa B (NF-κB) signaling increases the expression of ARVs in prostate cancer (PCa) cells and converts androgen-sensitive PCa cells to become androgen-insensitive, whereas downregulation of NF-κB signaling inhibits ARV expression and restores responsiveness of CRPC to anti-androgen therapy. In addition, we demonstrated that combination of anti-androgen with NF-κB-targeted therapy inhibits efficiently tumor growth of human CRPC xenografts. These results indicate that induction of ARVs by activated NF-κB signaling in PCa cells is a critical mechanism by which the PCa progresses to CRPC. This has important implications as it can prolong the survival of CRPC patients by restoring the tumors to once again respond to conventional androgen-deprivation therapy (ADT). [ABSTRACT FROM AUTHOR]

Tissue inhibitor of metalloproteinases-1 (TIMP-1) recently emerged as a pro-metastatic factor highly associated with poor prognosis in a number of cancers. This correlation seemed paradox as TIMP-1 is best described as an inhibitor of pro-tumourigenic matrix metalloproteinases. Only recently, TIMP-1 has been revealed as a signalling molecule that can regulate cancer progression independent of its inhibitory properties. In the present study, we demonstrate that an increase of both exogenous and endogenous TIMP-1 led to the upregulation of miR-210 in a CD63/PI3K/AKT/HIF-1-dependent pathway in lung adenocarcinoma cells. TIMP-1 induced P110/P85 PI3K-signalling and AKT phosphorylation. It also led to increase of HIF-1α protein levels positively correlating with HIF-1-regulated mRNA expression and upregulation of the microRNA miR-210. Downstream targets of miR-210, namely FGFRL1, E2F3, VMP-1, RAD52 and SDHD, were decreased in the presence of TIMP-1. Upon the overexpression of TIMP-1 in tumour cells, miR-210 was accumulated in exosomes in vitro and in vivo. These exosomes promoted tube formation activity in human umbilical vein endothelial cell (HUVECs), which was reflected in increased angiogenesis in A549L-derived tumour xenografts. Activation and elevation of PI3K, AKT, HIF-1A and miR-210 in tumours additionally confirmed our in vitro data. This new pro-tumourigenic signalling function of TIMP-1 may explain why elevated TIMP-1 levels in lung cancer patients are highly correlated with poor prognosis. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homolog (PTEN), which negatively regulates tumorigenic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling, is a commonly mutated tumor suppressor. The majority of cancer-associated PTEN mutations block its essential PIP3 phosphatase activity. However, there is a group of clinically identified PTEN mutations that maintain enzymatic activity, and it is unknown how these mutations contribute to tumor pathogenesis. Here, we show that these enzymatically competent PTEN mutants fail to translocate to the plasma membrane where PTEN converts PIP3 to PI(4,5)P2. Artificial membrane tethering of the PTEN mutants effectively restores tumor suppressor activity and represses excess PIP3 signaling in cells. Thus, our findings reveal a novel mechanism of tumorigenic PTEN deficiency. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is required for faithful repair of double-strand DNA breaks. Defects in HR repair cause severe genomic instability and challenge cellular viability. Paradoxically, various cancers are HR defective and have apparently acquired characteristics to survive genomic instability. We aimed to identify these characteristics to uncover therapeutic targets for HR-deficient cancers. Cytogenetic analysis of 1143 ovarian cancers showed that the degree of genomic instability was correlated to amplification of replication checkpoint genes ataxia telangiectasia and Rad3-related kinase (ATR) and CHEK1. To test whether genomic instability leads to increased reliance on replication checkpoint signaling, we inactivated Rad51 to model HR-related genomic instability. Rad51 inactivation caused defective HR repair and induced aberrant replication dynamics. Notably, inhibition of Rad51 led to increased ATR/checkpoint kinase-1 (Chk1)-mediated replication stress signaling. Importantly, inhibition of ATR or Chk1 preferentially killed HR-deficient cancer cells. Combined, our data show that defective HR caused by Rad51 inhibition results in differential sensitivity for ATR and Chk1 inhibitors, implicating replication checkpoint kinases as potential drug targets for HR-defective cancers. [ABSTRACT FROM AUTHOR]

We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/β-catenin signaling directly regulates FGF1 expression via TCF binding elements in the FGF1-1C promoter locus. In vitro gene manipulation studies revealed that FGF1 is sufficient to drive the tumor-promoting effects of WNT7A. In vivo xenograft studies confirmed that the stable overexpression of WNT7A or FGF1 induced a significant increase in tumor incidence, whereas FGF1 knockdown in WNT7A overexpressing cells caused a significant reduction in tumor size. Niclosamide most efficiently abrogated WNT7A/β-catenin signaling in our model, inhibited β-catenin transcriptional activity and cell viability, and increased cell death. Furthermore, niclosamide decreased cell migration following an increase in E-cadherin subsequent to decreased levels of SLUG. The effects of niclosamide on cell functions were more potent in WNT7A-overexpressing cells. Oral niclosamide inhibited tumor growth and progression in an intraperitoneal xenograft mouse model representative of human ovarian cancer. Collectively, these results indicate that FGF1 is a direct downstream target of WNT7A/β-catenin signaling and this pathway has potential as a therapeutic target in ovarian cancer. Moreover, niclosamide is a promising inhibitor of this pathway and may have clinical relevance. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3. [ABSTRACT FROM AUTHOR]

Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAFV637E knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation. [ABSTRACT FROM AUTHOR]

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration. [ABSTRACT FROM AUTHOR]

The DEAD-box RNA helicase DDX3 is a versatile protein involved in multiple steps of gene expression and various cellular signaling pathways. DDX3 mutations have been implicated in the wingless (Wnt) type of medulloblastoma. We show here that small interfering RNA-mediated DDX3 knockdown in various cell lines increased cell-cell adhesion but decreased cell-extracellular matrix adhesion. Moreover, DDX3 depletion suppressed cell motility and impaired directional migration in the wound-healing assay. Accordingly, DDX3-depleted cells exhibited reduced invasive capacities in vitro as well as reduced metastatic potential in mice. We also examined the mechanism underlying DDX3-regulated cell migration. DDX3 knockdown reduced the levels of both Rac1 and β-catenin proteins, and consequentially downregulated the expression of several β-catenin target genes. Moreover, we demonstrated that DDX3-regulated Rac1 mRNA translation, possibly through an interaction with its 5′-untranslated region, and affected β-catenin protein stability in an Rac1-dependent manner. Taken together, our results indicate the DDX3-Rac1-β-catenin regulatory axis in modulating the expression of Wnt/β-catenin target genes. Therefore, this report provides a mechanistic context for the role of DDX3 in Wnt-type tumors. [ABSTRACT FROM AUTHOR]

Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of Wnt signaling pathway. We aim to clarify its epigenetic inactivation, biological function and clinical implication in colon cancer. DACT2 was silenced in five out of eight colon cancer cell lines, but robustly expressed in normal colon tissues. The loss of DACT2 expression was regulated by promoter hypermethylation. Restoring DACT2 expression in colon cancer cell lines suppressed tumor cell growth by inducing cell apoptosis and inhibiting cell proliferation both in vitro and in vivo. Moreover, DACT2 overexpression effectively reduced lung metastasis of colon cancer cells in nude mice. These effects by DACT2 were attributed to inhibition of Wnt/β-catenin signaling. Reexpression of DACT2 significantly suppressed the transcriptional activity of both wild-type β-catenin and degradation-resistant form mutant β-catenin (S33Y). DACT2 could actively shuttle into and out of nuclei, with its predominant steady-state localization in the cytoplasm dependent on its nuclear export signal. Co-immunoprecipitation results indicated that DACT2 strongly associated β-catenin as well as lymphoid enhancer-binding factor 1 (LEF1) and directly disrupted the formation of the β-catenin-LEF1 complex in the nucleus. Whereas in the cytoplasm, DACT2 restored junctional localization of E-cadherin-β-catenin complexes and prevented β-catenin nuclear translocation through direct interaction with β-catenin. DACT2 methylation was detected in 43.3% (29/67) of colon cancer tissues, but none in normal controls. Multivariate analysis revealed that patients with DACT2 methylation had a significant decrease in overall survival (P=0.006). Kaplan-Meier survival curves showed that DACT2 methylation was significantly associated with shortened survival in stage I-III colon cancer patients. In conclusion, DACT2 acts as a functional tumor suppressor in colon cancer through inhibiting Wnt/β-catenin signaling. Its methylation at early stages of colon carcinogenesis is an independent prognostic factor. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are secreted cytokines/growth factors that have differing roles in cancer. BMPs are overexpressed in human breast cancers, but loss of BMP signaling in mammary carcinomas can accelerate metastasis. We show that human breast cancers display active BMP signaling, which is rarely downregulated or homozygously deleted. We hypothesized that systemic inhibition of BMP signaling in both the tumor and the surrounding microenvironment could prevent tumor progression and metastasis. To test this hypothesis, we used DMH1, a BMP antagonist, in MMTV.PyVmT expressing mice. Treatment with DMH1 reduced lung metastasis and the tumors were less proliferative and more apoptotic. In the surrounding tumor microenvironment, treatment with DMH1 altered fibroblasts, lymphatic vessels and macrophages to be less tumor promoting. These results indicate that inhibition of BMP signaling may successfully target both the tumor and the surrounding microenvironment to reduce tumor burden and metastasis. [ABSTRACT FROM AUTHOR]

The TP53 tumor-suppressor gene is frequently mutated in human cancer. Missense mutations can add novel functions (gain-of-function, GOF) that promote tumor malignancy. Here we report that mutant (mut) p53 promotes tumor malignancy by suppressing the expression of a natural occurring anti-inflammatory cytokine, the secreted interleukin-1 receptor antagonist (sIL-1Ra, IL1RN). We show that mutp53 but not wild-type (wt) p53 suppresses the sIL-1Ra production in conditioned media of cancer cells. Moreover, mutp53, but not wtp53, binds physically the sIL-1Ra promoter and the protein-protein interaction with the transcriptional co-repressor MAFF (v-MAF musculoaponeurotic fibrosarcoma oncogene family, protein F) is required for mutp53-induced sIL-1Ra suppression. Remarkably, when exposed to IL-1 beta (IL-1β) inflammatory stimuli, mutp53 sustains a ready-to-be-activated in vitro and in vivo cancer cells' response through the sIL-1Ra repression. Taken together, these results identify sIL-1Ra as a novel mutp53 target gene, whose suppression might be required to generate a chronic pro-inflammatory tumor microenvironment through which mutp53 promotes tumor malignancy. [ABSTRACT FROM AUTHOR]

CYLD is a deubiquitinating (DUB) enzyme that has a pivotal role in modulating nuclear factor kappa B (NF-κB) signaling pathways by removing the lysine 63- and linear-linked ubiquitin chain from substrates such as tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Loss of CYLD activity is associated with tumorigenicity, and levels of CYLD are lost or downregulated in different types of human tumors. In the present study, we found that high CYLD expression was associated with better overall survival and relapse-free neuroblastoma patient outcome, as well as inversely correlated with the stage of neuroblastoma. Retinoic acid-mediated differentiation of neuroblastoma restored CYLD expression and promoted SUMOylation of CYLD. This posttranslational modification inhibited deubiquitinase activity of CYLD against TRAF2 and TRAF6 and facilitated NF-κB signaling. Overexpression of non-SUMOylatable mutant CYLD in neuroblastoma cells reduced retinoic acid-induced NF-κB activation and differentiation of cells, but instead promoted cell death. [ABSTRACT FROM AUTHOR]

The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-β) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitioned (EMT) phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-β pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-β signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor. [ABSTRACT FROM AUTHOR]

Kinase suppressor of Ras-1 (KSR1) facilitates signal transduction in Ras-dependent cancers, including pancreatic and lung carcinomas but its role in breast cancer has not been well studied. Here, we demonstrate for the first time it functions as a tumor suppressor in breast cancer in contrast to data in other tumors. Breast cancer patients (n>1000) with high KSR1 showed better disease-free and overall survival, results also supported by Oncomine analyses, microarray data (n=2878) and genomic data from paired tumor and cell-free DNA samples revealing loss of heterozygosity. KSR1 expression is associated with high breast cancer 1, early onset (BRCA1), high BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels. Phospho-profiling of major components of the canonical Ras-RAF-mitogen-activated protein kinases pathway showed no significant changes after KSR1 overexpression or silencing. Moreover, KSR1 stably transfected cells formed fewer and smaller size colonies compared to the parental ones, while in vivo mouse model also demonstrated that the growth of xenograft tumors overexpressing KSR1 was inhibited. The tumor suppressive action of KSR1 is BRCA1 dependent shown by 3D-matrigel and soft agar assays. KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance. These data link these proteins in a continuum with clinical relevance and position KSR1 in the major oncoprotein pathways in breast tumorigenesis. [ABSTRACT FROM AUTHOR]

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1S425D led to increased β1 integrin activation and generated biologic effects opposite to talin1S425A expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1S425D restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells. [ABSTRACT FROM AUTHOR]

Mutations of Wnt/β-catenin signaling pathway has essential roles in development and cancer. Although β-catenin and adenomatous polyposis coli (APC) gene mutations are well established and are known to drive tumorigenesis, discoveries of mutations in other components of the pathway lagged, which hinders the understanding of cancer mechanisms. Here we report that δ-catenin (gene designation: CTNND2), a primarily neural member of the β-catenin superfamily that promotes canonical Wnt/β-catenin/LEF-1-mediated transcription, displays exonic mutations in human prostate cancer and promotes cancer cell survival adaptation and metabolic reprogramming. When overexpressed in cells derived from prostate tumor xenografts, δ-catenin gene invariably gives rise to mutations, leading to sequence disruptions predicting functional alterations. Ectopic δ-catenin gene integrating into host chromosomes is locus nonselective. δ-Catenin mutations promote tumor development in mouse prostate with probasin promoter (ARR2PB)-driven, prostate-specific expression of Myc oncogene, whereas mutant cells empower survival advantage upon overgrowth and glucose deprivation. Reprogramming energy utilization accompanies the downregulation of glucose transporter-1 and poly (ADP-ribose) polymerase cleavage while preserving tumor type 2 pyruvate kinase expression. δ-Catenin mutations increase β-catenin translocation to the nucleus and hypoxia-inducible factor 1α (HIF-1α) expression. Therefore, introducing δ-catenin mutations is an important milestone in prostate cancer metabolic adaptation by modulating β-catenin and HIF-1α signaling under glucose shortage to amplify its tumor-promoting potential. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS), a neoplasm characterised by undifferentiated myoblasts, is the most common soft tissue tumour of childhood. Although aggressive treatment of RMS could provide long-term benefit, resistance to current therapies is an ongoing problem. We report here that insulin-like growth factor 2-binding protein 1 (IGF2BP1), an oncofetal protein, is expressed in RMS patient-derived cell lines and in primary tumours where it drives translation of the cellular inhibitor of apoptosis 1 (cIAP1), a key regulator of the nuclear factor-κB signalling pathway and of caspase-8-mediated cell death. We demonstrate that reducing the levels of cIAP1 in RMS, either by IGF2BP1 knockdown or by IAP antagonists, sensitises these cells to tumour necrosis factor-α-mediated cell death. Finally, we show that targeting cIAP1 by IAP antagonists delays RMS tumour growth and improve survival in mice. Our results identify IGF2BP1 as a critical translational regulator of cIAP1-mediated apoptotic resistance in RMS and advocate for the combined use of IAP antagonists and tumour necrosis factor-α as a therapeutic approach for this type of cancer. [ABSTRACT FROM AUTHOR]

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers. [ABSTRACT FROM AUTHOR]

Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans. [ABSTRACT FROM AUTHOR]

HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody target. In this study, we identified NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel interaction partner and ubiquitin E3 ligase of human HER3. Using molecular and biochemical approaches, we demonstrated that the C-terminal tail of HER3 interacted with the WW domains of NEDD4 and the interaction was independent of neuregulin-1. Short hairpin RNA knockdown of NEDD4 elevated HER3 levels and resulted in increased HER3 signaling and cancer cell proliferation in vitro and in vivo. A similar inverse relationship between HER3 and NEDD4 levels was observed in prostate cancer tumor tissues. More importantly, the upregulated HER3 expression by NEDD4 knockdown sensitized cancer cells for growth inhibition by an anti-HER3 antibody. Taken together, our results suggest that low NEDD4 levels may predict activation of HER3 signaling and efficacies of anti-HER3 antibody therapies. [ABSTRACT FROM AUTHOR]

Extracellular proteins are vital for cell activities, such as cell migration. Calumenin is highly conserved among eukaryotes, but its functions are largely unclear. Here, we identify extracellular calumenin as a suppressor of cell migration and tumor metastasis. Calumenin binds to and stabilizes fibulin-1, leading to inactivation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling. We further identify the minimal functional domain of calumenin (amino acids 74-138 and 214-280). Depletion of calumenin induces fibulin-1- and phospho-ERK1/2 (pERK1/2)-dependent promotion of cell migration. Consistently, in hepatocellular and pancreatic carcinoma, both calumenin and fibulin-1 are downregulated. Furthermore, we show that matrix metalloproteinase-13 (MMP-13) proteolyzes fibulin-1 and that calumenin protects fibulin-1 from cleavage by MMP-13. Calumenin, together with fibulin-1, also interacts with fibronectin and depends on both syndecan-4 and α5β1-integrin to suppress ERK1/2 signaling and inhibit cell migration. Thus, extracellular calumenin regulates fibulin-1 to have crucial roles in ERK1/2 signaling and cell migration. [ABSTRACT FROM AUTHOR]

High cellular heterogeneity within neuroblastomas (NBs) may account for the non-uniform response to treatment. c-KIT+ cells are frequently detected in NB, but how they influence NB behavior still remains elusive. Here, we used NB tumor-initiating cells to reconstitute NB development and demonstrated that c-KIT+ cells are de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIT+ NB cells express higher levels of neural crest and stem cell markers (SLUG, SOX2 and NANOG) and are endowed with high clonogenic capacity, differentiation plasticity and are refractory to drugs. With serial transplantation assays, we found that c-KIT expression is not required for tumor formation, but c-KIT+ cells are more aggressive and can induce tumors ninefold more efficiently than c-KIT−/low cells. Intriguingly, c-KIT+ cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of secondary and tertiary tumors in mice. In addition, we showed that Prokineticin signaling and mitogen-activated protein kinase pathways are crucial for the maintenance of c-KIT+ cells in tumor to promote NB progression. Our results highlight the importance of this de novo population of NB cells in sustainable growth of NB and reveal specific signaling pathways that may provide targets leading to more effective NB therapies. [ABSTRACT FROM AUTHOR]

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using 125I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca2+-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding. [ABSTRACT FROM AUTHOR]

In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies. [ABSTRACT FROM AUTHOR]

Sirtuins are class III deacetylases that regulate many essential processes, including cellular stress, genome stability and metabolism. Although these NAD+-dependent deacetylases control adaptive cellular responses, identification of sirtuin-regulated signaling targets remain under-studied. Here, we demonstrate that acetylation of the mitogen-activated protein kinase kinase-1 (MEK1) stimulates its kinase activity, and that acetylated MEK1 is under the regulatory control of the sirtuin family members SIRT1 and SIRT2. Treatment of cells with sirtuin inhibitors, or siRNA knockdown of SIRT1 or SIRT2 proteins, increases MEK1 acetylation and subsequent phosphorylation of the extracellular signal-regulated kinase. Generation of an acetyl-specific MEK1 antibody demonstrates that endogenous acetylated MEK1 is extensively enriched in the nucleus following epidermal growth factor stimulation. An acetyl-mimic of MEK1 increases inappropriate growth properties, suggesting that acetylation of MEK1 has oncogenic potential. [ABSTRACT FROM AUTHOR]

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvβ6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFβ1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvβ6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFβ pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvβ6-dependent, once fibroblasts are activated alternative TGFβ pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvβ6 expression, activate normal fibroblasts through avβ6/TGFβ signalling in vitro, and influence both gene expression and response to therapeutic agents. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection. [ABSTRACT FROM AUTHOR]

The tripartite motif protein TRIM24 (tripartite motif-containing 24) has been found to play distinct roles in tumor development and progression, according to different tumor contexts. However, it remains elusive whether TRIM24 plays a role in malignant gliomas that are the most common and deadly primary brain tumors in adults. We report here that TRIM24 expression is positively correlated with glioma malignancy and is negatively associated with prognosis of patients with newly diagnosed glioblastoma, which is the most malignant form of gliomas but displays highly heterogeneous clinical outcome. The multivariate Cox regression analysis demonstrates the independent predictive value of TRIM24 expression level for overall and progression-free survival. Knockdown of TRIM24 suppresses cell proliferation, cell cycle progression, clone formation and in vivo tumor development, whereas overexpression of TRIM24 promotes cell growth. Chromatin immunoprecipitation, real-time reverse transcription-PCR and mutation analyses demonstrate that TRIM24 binds to the PIK3CA promoter via its PHD-Bromo domain to activate the transcription of PIK3CA gene, thus enhancing phosphatidylinositide 3-kinase (PI3K)/Akt signaling. The pan-PI3K inhibitor LY294002 and small interfering RNA targeting PIK3CA both abrogate the growth-promoting effect of TRIM24. Moreover, TRIM24 regulates the expression of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) through PI3K/Akt/nuclear factor-κB signaling transduction and enhances resistance to temozolomide, the standard chemotherapeutic agent for glioblastoma. Finally, glioblastoma patients with low TRIM24 expression benefit from chemotherapy, whereas those with high TRIM24 expression do not have such benefit. Our results suggest that TRIM24 might serve as a potential prognostic marker and therapeutic target for the management of malignant gliomas. [ABSTRACT FROM AUTHOR]