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In psychotherapy, paradoxical interventions are characterized by a deliberate reinforcement of the pathological behavior to improve the clinical condition. Such a counter‐intuitive approach can be considered when more conventional interventions fail. The development of targeted cancer therapies has enabled the selective inhibition of activated oncogenic signaling pathways. However, in advanced cancers, such therapies, on average, deliver modest benefits due to the development of resistance. Here, we review the perspective of a ‘paradoxical intervention’ in cancer therapy: rather than attempting to inhibit oncogenic signaling, the proposed therapy would further activate mitogenic signaling to disrupt the labile homeostasis of cancer cells and overload stress response pathways. Such overactivation can potentially be combined with stress‐targeted drugs to kill overstressed cancer cells. Although counter‐intuitive, such an approach exploits intrinsic and ubiquitous differences between normal and cancer cells. We discuss the background underlying this unconventional approach and how such intervention might address some current challenges in cancer therapy.

BackgroundUnderstanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFβ or activin-induced metastatic phenotype of colon cancer.MethodMitogenic signaling/growth factor receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (activin receptor 2) -/- or wild type mice. Colon cancer cell lines (+/- SMAD4) were interrogated for ligand-induced PI3K and MEK/ERK pathway activation and downstream protein/phospho-isoform expression/association after knockdown and pharmacologic inhibition of pathway members. EMT was assessed using epithelial/mesenchymal markers and migration assays.ResultsIn primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 expression was associated with TGFβ/MEK/ERK pathway activation. Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with activin-dependent PI3K signaling.ConclusionAlthough activin and TGFβ share growth suppressive SMAD signaling in colon cancer, they diverge in their SMAD4-independent pro-migratory signaling utilizing distinct mitogenic signaling pathways that affect EMT. p21 localization in colon cancer may determine a dominant activin versus TGFβ ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFβ family receptors.

In psychotherapy, paradoxical interventions are characterized by a deliberate reinforcement of the pathological behavior to improve the clinical condition. Such a counter‐intuitive approach can be considered when more conventional interventions fail. The development of targeted cancer therapies has enabled the selective inhibition of activated oncogenic signaling pathways. However, in advanced cancers, such therapies, on average, deliver modest benefits due to the development of resistance. Here, we review the perspective of a 'paradoxical intervention' in cancer therapy: rather than attempting to inhibit oncogenic signaling, the proposed therapy would further activate mitogenic signaling to disrupt the labile homeostasis of cancer cells and overload stress response pathways. Such overactivation can potentially be combined with stress‐targeted drugs to kill overstressed cancer cells. Although counter‐intuitive, such an approach exploits intrinsic and ubiquitous differences between normal and cancer cells. We discuss the background underlying this unconventional approach and how such intervention might address some current challenges in cancer therapy. [ABSTRACT FROM AUTHOR]

In malignant transformation, cellular stress‐response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress‐targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K‐Ras‐driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response‐checkpoint inhibitors triggered cell death. CRISPR/Cas9‐mediated K‐Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE‐821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK‐ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress‐targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies.

In malignant transformation, cellular stress‐response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress‐targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K‐Ras‐driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response‐checkpoint inhibitors triggered cell death. CRISPR/Cas9‐mediated K‐Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE‐821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK‐ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress‐targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies. We designed an unexplored approach to cancer therapy: mitogenic overstimulation to increase dependence on stress‐response pathways, plus stress‐targeted inhibitors to selectively kill cancer cells. As a proof of principle, we show that exogenous FGF2 enhanced replication and proteotoxic stresses in different cancer cell lines, rendering these cells prone to massive cell death when combined with ATR or proteasome inhibitors. [ABSTRACT FROM AUTHOR]

Abstract Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment. Highlights • PTHrP activates ERK pathway through PKC, Scr and Akt in CRC cells. • PTHrP induces resistance to Irinotecan in Caco-2 and HCT116 cells. • The chemoresistance by PTHrP in CRC cells is mediated through ERK. • PTHrP administration increases ERK protein levels in nude mice xenografts. • In vivo PTHrP modulates markers related to tumorigenic events of CRC. [ABSTRACT FROM AUTHOR]

Since Src kinase inhibitors decrease brain injury produced by intracerebral hemorrhage (ICH) and thrombin is activated following ICH, this study determined whether Src kinase inhibitors decrease thrombin-induced brain injury. Thrombin injections into adult rat striatum produced focal infarction and motor deficits. The Src kinase inhibitor PP2 decreased thrombin-induced Src activation, infarction in striatum and motor deficits in vivo. Thrombin applied to cultured post-mitotic striatal neurons caused: injury to axons and dendrites; many TUNEL positive neuronal nuclei; and re-entry into the cell cycle as manifested by cyclin D1 expression, induction of several other cell cycle genes and cyclin-dependent kinase 4 activation. PP2 dose-dependently attenuated thrombin-induced injury to the cultured neurons; and attenuated thrombin-induced neuronal cell cycle re-entry. These results are consistent with the hypotheses that Src kinase inhibitors decrease injury produced by ICH by decreasing thrombin activation of Src kinases and, at least in part, by decreasing Src induced cell cycle re-entry.

Neurons that reenter the cell cycle die rather than divide, a phenomenon that is associated with neurodegeneration in Alzheimer's disease (AD). Reexpression of cell-cycle related genes in differentiated neurons in AD might be rooted in aberrant mitogenic signaling. Because microglia and astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque components or factors secreted from plaque-activated glia induce neuronal mitogenic signaling. Advanced glycation end products (AGEs), protein-bound oxidation products of sugar, might be one of those mitogenic compounds. Cyclin D1 positive neurons are colocalized with AGEs or directly surrounded by extracellular AGE deposits in AD brain. However, a direct proof of DNA replication in these cells has been missing. Here, we report by using fluorescent in situ hybridization that consistent with the expression of cell cycle proteins, hyperploid neuronal cells are in colocalization with AGE staining in AD brains but not in nondemented controls. To complement human data, we used apolipoprotein E-deficient mice as model of neurodegeneration and showed that increased oxidative stress caused an intensified neuronal deposition of AGEs, being accompanied by an activation of the MAPK cascade via RAGE. This cascade, in turn, induced the expression of cyclin D1 and DNA replication. In addition, reduction of oxidative stress by application of α-lipoic acid decreased AGE accumulations, and this decrease was accompanied by a reduction in cell cycle reentry and a more euploid neuronal genome. [ABSTRACT FROM AUTHOR]

Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly ( p < 0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro-angiogenic (VEGF, iNOS), migratory (MMP-2) and EMT promoting proteins (uPA, vimentin, N-cadherin) were up-regulated by IR in PCa cells. Interestingly, all of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR-activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3, by silibinin. [ABSTRACT FROM AUTHOR]

Rit, a member of the Ras family of GTPases, has been shown to promote cell survival in response to oxidative stress, in part by directing an evolutionarily conserved p38 MAPK-Akt survival cascade. Aberrant Rit signaling has recently been implicated as a driver mutation in human cancer, adding importance to the characterization of critical Rit effector pathways. However, the mechanism by which Rit-p38 signaling regulated Akt activity was unknown. Here, we identify mTORC2 as a critical downstream mediator of Rit-dependent survival signaling in response to reactive oxygen species (ROS) stress. Rit interacts with Sin1 (MAPKAP1), and Rit loss compromises ROS-dependent mTORC2 complex activation, blunting mTORC2-mediated phosphorylation of Akt kinase. Taken together, our findings demonstrate that the p38/mTORC2/Akt signaling cascade mediates Rit-dependent oxidative stress survival. Inhibition of this previously unrecognized cascade should be explored as a potential therapy of Rit-dependent malignancies. [ABSTRACT FROM AUTHOR]

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways. [ABSTRACT FROM AUTHOR]

The ATP-gated P2X7 has been shown to play an important role in invasiveness and metastasis of some tumors. However, the possible links and underlying mechanisms between P2X7 and prostate cancer have not been elucidated. Here, we demonstrated that P2X7 was highly expressed in some prostate cancer cells. Down-regulation of P2X7 by siRNA significantly attenuated ATP- or BzATP-driven migration and invasion of prostate cancer cells in vitro, and inhibited tumor invasiveness and metastases in nude mice. In addition, silencing of P2X7 remarkably attenuated ATP- or BzATP- driven expression changes of EMT/invasion-related genes Snail, E-cadherin, Claudin-1, IL-8 and MMP-3, and weakened the phosphorylation of PI3K/AKT and ERK1/2 in vitro. Similar effects were observed in nude mice. These data indicate that P2X7 stimulates cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes, as well as PI3K/AKT and ERK1/2 signaling pathways. P2X7 could be a promising therapeutic target for prostate cancer. [ABSTRACT FROM AUTHOR]

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication. [ABSTRACT FROM AUTHOR]

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. [ABSTRACT FROM AUTHOR]

A high-fat diet coincides with increased levels of bile acids. This increase in bile acids, particularly deoxycholic acid (DCA), has been strongly associated with the development of colon cancer. Conversely, ursodeoxycholic acid (UDCA) may have chemopreventive properties. Although structurally similar, DCA and UDCA present different biological and pathological effects in colon cancer progression. The differential regulation of cancer by these two bile acids is not yet fully understood. However, one possible explanation for their diverging effects is their ability to differentially regulate signaling pathways involved in the multistep progression of colon cancer, such as the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway. This review will examine the biological effects of DCA and UDCA on colon cancer development, as well as the diverging effects of these bile acids on the oncogenic signaling pathways that play a role in colon cancer development, with a particular emphasis on bile acid regulation of the EGFR-MAPK pathway. [ABSTRACT FROM AUTHOR]

Colletotrichum orbiculare is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP–PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1–Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP–CoRAS2. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2. [ABSTRACT FROM AUTHOR]

Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion. [ABSTRACT FROM AUTHOR]

Background and Aims: Endoplasmic reticulum (ER) stress is induced in many forms of chronic liver disease and may promote the development of hepatocellular carcinoma. The activator protein 1 (AP-1) complex is a transcription factor that promotes hepatic carcinogenesis in response to cellular stress. The aim of this study was to determine the role of ER stress in the regulation of the hepatic AP-1 complex. Methods: Human hepatocellular carcinoma (HepG2) cells and C57BL/6J mice were subjected to pharmacologic ER stress and the expression of AP-1-associated genes and proteins was assessed. To determine the role of MAPK signaling in ER stress-induced AP-1 activation, ER stress was induced in JNK- and ERK-inhibited HepG2 cells. Results: Induction of ER stress promoted the activation of both Jun- and Fos-related genes and proteins of the AP-1 complex in HepG2 cells and murine liver. Inhibition of ERK phosphorylation in HepG2 cells completely prevented ER stress-induced activation of the fos-related components of AP-1 whereas activation of Jun-related components was only partially attenuated. Conversely, inhibition of JNK phosphorylation in HepG2 cells reduced ER stress-induced activation of Jun-related components but did not prevent activation of fos-related components. Conclusions: ER stress activates the hepatic AP-1 complex via MAPK-dependent signaling pathways. ER stress-induced activation of Fos-related components is dependent primarily on ERK activation whereas ER stress-induced activation of Jun-related components is dependent primarily on JNK activation, although there is interplay between these regulatory pathways. These data implicate a novel signaling pathway by which sustained ER stress, as observed in many chronic liver diseases, may promote hepatic carcinogenesis. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation. [ABSTRACT FROM AUTHOR]

KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-κB-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-α and IFN-γ, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 µg/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and apoptosis via activation of JNK signaling pathways, and KIOM-C represents a safe and potent herbal therapy for treating malignancies. [ABSTRACT FROM AUTHOR]

Few of >150 published cell cycle modeling efforts use significant levels of data for tuning and validation. This reflects the difficultly to generate correlated quantitative data, and it points out a critical uncertainty in modeling efforts. To develop a data-driven model of cell cycle regulation, we used contiguous, dynamic measurements over two time scales (minutes and hours) calculated from static multiparametric cytometry data. The approach provided expression profiles of cyclin A2, cyclin B1, and phospho-S10-histone H3. The model was built by integrating and modifying two previously published models such that the model outputs for cyclins A and B fit cyclin expression measurements and the activation of B cyclin/Cdk1 coincided with phosphorylation of histone H3. The model depends on Cdh1-regulated cyclin degradation during G1, regulation of B cyclin/Cdk1 activity by cyclin A/Cdk via Wee1, and transcriptional control of the mitotic cyclins that reflects some of the current literature. We introduced autocatalytic transcription of E2F, E2F regulated transcription of cyclin B, Cdc20/Cdh1 mediated E2F degradation, enhanced transcription of mitotic cyclins during late S/early G2 phase, and the sustained synthesis of cyclin B during mitosis. These features produced a model with good correlation between state variable output and real measurements. Since the method of data generation is extensible, this model can be continually modified based on new correlated, quantitative data. [ABSTRACT FROM AUTHOR]

Heat shock factor 1 (HSF1) is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth. [ABSTRACT FROM AUTHOR]

The chaperone nucleophosmin (NPM1) is over-expressed in the epithelial compartment of prostate tumours compared to adjacent healthy epithelium and may represent one of the key actors that support the neoplastic phenotype of prostate adenocarcinoma cells. Yet, the mechanisms that underlie NPM1 mediated phenotype remain elusive in the prostate. To better understand NPM1 functions in prostate cancer cells, we sought to characterize its impact on prostate cancer cells behaviour and decipher the mechanisms by which it may act. Here we show that NPM1 favors prostate tumour cell migration, invasion and colony forming. Furthermore, knockdown of NPM1 leads to a decrease in the growth of LNCaP-derived tumours grafted in Nude mice in vivo. Such oncogenic-like properties are found in conjunction with a positive regulation of NPM1 on the ERK1/2 (Extracellular signal-Regulated Kinases 1/2) kinase phosphorylation in response to EGF (Epidermal Growth Factor) stimulus, which is critical for prostate cancer progression following the setting of an autonomous production of the growth factor. NPM1 could then be a target to switch off specifically ERK1/2 pathway activation in order to decrease or inhibit cancer cell growth and migration. [ABSTRACT FROM AUTHOR]

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior. [ABSTRACT FROM AUTHOR]

Background/Aim: Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved. Methods: Cultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed. Results: In presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group. Conclusion: Sodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs. [ABSTRACT FROM AUTHOR]

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma. [ABSTRACT FROM AUTHOR]

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation. [ABSTRACT FROM AUTHOR]

Abstract: Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5μM), and anti-proliferative and toxic at high concentration (100μM). Low H2O2 concentration triggered activation of nSMase2 and SK1 through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFRβ, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SK1. The H2O2-induced src/PDGFRβ/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFRβ/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress. [Copyright &y& Elsevier]

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3Ras mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner. [ABSTRACT FROM AUTHOR]

Stimulation of α1aAdrenergic Receptors (ARs) is known to have anti-proliferative and hypertrophic effects; however, some studies also suggests this receptor can increase cell proliferation. Surprisingly, we find the α1aAR expressed in rat-1 fibroblasts can produce either phenotype, depending exclusively on agonist concentration. Stimulation of the α1aAR by high dose phenylephrine (>10−7 M) induces an antiproliferative, hypertrophic response accompanied by robust and extended p38 activation. Inhibition of p38 with SB203580 prevented the antiproliferative response, while inhibition of Erk or Jnk had no effect. In stark contrast, stimulation of the α1aAR with low dose phenylephrine (∼10−8 M) induced an Erk-dependent increase in cellular proliferation. Agonist-induced Erk phosphorylation was preceded by rapid FGFR and EGFR transactivation; however, only EGFR inhibition blocked Erk activation and proliferation. The general matrix metalloprotease inhibitor, GM6001, blocked agonist induced Erk activation within seconds, strongly suggesting EGFR activation involved extracellular triple membrane pass signaling. Erk activation required little Ca2+ release and was blocked by PLCβ or PKC inhibition but not by intracellular Ca2+ chelation, suggesting Ca2+ independent activation of novel PKC isoforms. In contrast, Ca2+ release was essential for PI3K/Akt activation, which was acutely maximal at non-proliferative doses of agonist. Remarkably, our data suggests EGFR transactivation leading to Erk induced proliferation has the lowest activation threshold of any α1aAR response. The ability of α1aARs to induce proliferation are discussed in light of evidence suggesting antagonistic growth responses reflect native α1aAR function. [ABSTRACT FROM AUTHOR]

Endocrine resistance and metastatic progression are primary causes of treatment failure in breast cancer. While mitogen activated protein kinases (MAPKs) are known to promote ligand-independent cell growth, the role of the MEK5-ERK5 pathway in the progression of clinical breast carcinoma remains poorly understood. Here, we demonstrated increased ERK5 activation in 30 of 39 (76.9%) clinical tumor samples, as well as across breast cancer cell systems. Overexpression of MEK5 in MCF-7 cells promoted both hormone-dependent and hormone-independent tumorigenesis in vitro and in vivo and conferred endocrine therapy resistance to previously sensitive breast cancer cells. Expression of MEK5 suppressed estrogen receptor (ER)α, but not ER-β protein levels, and abrogated downstream estrogen response element (ERE) transcriptional activity and ER-mediated gene transcription. Global gene expression changes associated with upregulation of MEK5 included increased activation of ER-α independent growth signaling pathways and promotion of epithelial-to-mesenchymal transition (EMT) markers. Taken together, our findings show that the MEK5-ERK5 pathway mediates progression to an ER(−), mesenchymal and endocrine therapy resistant phenotype. Given the need for new clinical therapeutic targets, our results demonstrate the therapeutic potential of targeting the MEK5-ERK5 pathway in breast cancer. [ABSTRACT FROM AUTHOR]

Angiogenesis is a critical factor in the growth and dissemination of solid tumors. Indeed, tumor vasculature is abnormal and contributes to the development and spread of malignancies by creating a hostile microenvironment. The alternative SDF-1/CXCL12 receptor, CXCR7, is frequently and specifically expressed in tumor-associated vessels. In this study, we examine the role of endothelium-expressed CXCR7 in tumor vascular dysfunction by specifically examining the contribution of CXCR7 to endothelial cell (EC) proliferation. We demonstrate that CXCR7 expression is sufficient to drive post-confluent growth in EC cultures. Further, we provide a novel mechanism for CXCR7-mediated proliferation via proteasomal degradation of the tumor suppressor protein Rb. These findings identify a heretofore unappreciated role for CXCR7 in vascular dysfunction and confirm this receptor as a plausible target for anti-tumor therapy. [ABSTRACT FROM AUTHOR]

Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. [ABSTRACT FROM AUTHOR]

Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. [ABSTRACT FROM AUTHOR]

During rat postnatal development, gastric cell proliferation and differentiation depend on many elements, which include dietary pattern, hormones, growth factors and their signaling pathways. Among them, EGFR activity is increased through MAPK and Src cascades in response to early weaning that represents the abrupt transition from milk to solid food. We herein investigated the direct involvement of ERK pathway in the control of cell cycle progression during early weaning, and studied the specific role of p27. At 15 days, Wistar rats were separated from dams, fed with powdered chow and daily injected with PD98059 (MEK inhibitor, 300 µg/kg) or 0.5% DMSO (control). By using HE staining and immunohistochemistry for PCNA, we respectively detected mitotic (MI) and proliferative (PI) indices in 18-day-old pups, and observed that both were reduced by PD98059. As cell cycle-related proteins (cyclin E, CDK2, cyclin D1, CDK4, p21 and p27) are involved in proliferative regulation, we compared samples obtained at 17 days in the morning (17 d) and evening (17.5 d). We found that they were not altered after ERK inhibition, but cyclin D1, p21 and p27 levels changed throughout the day in the control group. As p27 activity depends on its integrity, we studied p27 phosphorylation (threonin 187), and observed that ERK inhibition reduced this process. We suggest that MAPK pathway interferes in the regulation of p27 function in the gastric mucosa during early weaning, possibly by controlling its degradation, and altogether this mechanism might contribute to the increase of epithelial proliferation at this condition. [ABSTRACT FROM AUTHOR]

Centrosome amplification (CA) is a contributor to carcinogenesis, generating aneuploidy, and chromosome instability. Previous work shows that breast adenocarcinomas have a higher frequency of centrosome defects compared to normal breast tissues. Abnormal centrosome phenotypes are found in pre-malignant lesions, suggesting an early role in breast carcinogenesis. However, the role of CA in breast cancers remains elusive. Identification of pathways and regulatory molecules involved in the generation of CA is essential to understanding its role in breast tumorigenesis. We established a breast cancer model of CA using Her2-positive cells. Our goal was to identify centrosome cycle molecules that are deregulated by aberrant Her2 signaling and the mechanisms driving CA. Our results show some Her2+ breast cancer cell lines harbor both CA and binucleation. Abolishing the expression of Cdk4 abrogated both CA and binucleation in these cells. We also found the source of binucleation in these cells to be defective cytokinesis that is normalized by downregulation of Cdk4. Protein levels of Nek2 diminish upon Cdk4 knockdown and vice versa, suggesting a molecular connection between Cdk4 and Nek2. Knockdown of Nek2 reduces CA and binucleation in this model while its overexpression further enhances centrosome amplification. We conclude that CA is modulated through Cdk4 and Nek2 signaling and that binucleation is a likely source of CA in Her2+ breast cancer cells. [ABSTRACT FROM AUTHOR]

Prolactin-induced Protein (PIP), an aspartyl protease unessential for normal mammalian cell function, is required for the proliferation and invasion of some breast cancer (BCa) cell types. Because PIP expression is particularly high in the Luminal A BCa subtype, we investigated the roles of PIP in the related T47D BCa cell line. Nucleic acid and antibody arrays were employed to screen effects of PIP silencing on global gene expression and activation of receptor tyrosine kinases (RTKs), respectively. Expression of PIP-stimulated genes, as defined in the T47D cell culture model, was well correlated with the expression of PIP itself across a cohort of 557 mRNA profiles of diverse BCa tumors, and bioinformatics analysis revealed cJUN and cMYC as major nodes in the PIP-dependent gene network. Among 71 RTKs tested, PIP silencing resulted in decreased phosphorylation of focal adhesion kinase (FAK), ephrin B3 (EphB3), FYN, and hemopoietic cell kinase (HCK). Ablation of PIP also abrogated serum-induced activation of the downstream serine/threonine kinases AKT, ERK1/2, and JNK1. Consistent with these results, PIP-depleted cells exhibited defects in adhesion to fibronectin, cytoskeletal stress fiber assembly and protein secretion. In addition, PIP silencing abrogated the mitogenic response of T47D BCa cells to estradiol (E2). The dependence of BCa cell proliferation was unrelated, however, to estrogen signaling because: 1) PIP silencing did not affect the transcriptional response of estrogen target genes to hormone treatment, and 2) PIP was required for the proliferation of tamoxifen-resistant BCa cells. Pharmacological inhibition of PIP may therefore serve the bases for both augmentation of existing therapies for hormone-dependent tumors and the development of novel therapeutic approaches for hormone-resistant BCa. [ABSTRACT FROM AUTHOR]

The 146-kDa Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the Gq/11-, G12/13- and Gi-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gαq/11. Subsequently, Gαq/11 activates RhoA via p63RhoGEF, which specifically interacts with Gαq/11 but not with other G proteins like Gα12/13 and Gαi. Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gαq/11-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida. [ABSTRACT FROM AUTHOR]

The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb. [ABSTRACT FROM AUTHOR]

Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK–activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21cip1. In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage. [ABSTRACT FROM AUTHOR]

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G0/G1 cell cycle inhibition (IC50 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27Kip1 expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27Kip1 expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis. [ABSTRACT FROM AUTHOR]

Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options. [ABSTRACT FROM AUTHOR]

Survivin is a unique member of the inhibitor of apoptosis (IAP) proteins that is overexpressed in numerous cancers through poorly defined mechanisms. One such mechanism may be through constitutive activation of the insulin-like growth factor-I (IGF-I) signaling pathway, implicated in the development and progression of prostate cancer. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that IGF-I induces Survivin expression, and that silencing Survivin by lentiviral-mediated small hairpin RNA (shRNA) represses IGF-I-stimulated cell growth, implicating Survivin as a mediator of this growth response. Moreover, our data support that the induction of Survivin by IGF-I occurs through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. Use of various Survivin promoter-luciferase constructs revealed that the CDE and CHR response elements in the proximal region of the Survivin promoter are involved in this IGF-I response. Transforming growth factor (TGF-β) signaling antagonists similarly activated the Surivin promoter and rendered cells refractory to further promoter activation by IGF-I. IGF-I suppressed levels of phospho-Smads 2 and 3 with kinetics similar to that of Survivin induction. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTOR. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin by IGF-I. Taken together, these results suggest that IGF-I signaling through a PI3K/Akt/mTORC1 mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling. [ABSTRACT FROM AUTHOR]

: Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis. [ABSTRACT FROM AUTHOR]

The Hippo (Hpo) pathway is a novel signaling pathway that controls organ size in Drosophila and mammals and is deregulated in a variety of human cancers. It consists of a set of kinases that, through a number of phosphorylation events, inactivate YAP, a transcriptional co-activator that controls cellular proliferation and apoptosis. We have identified PTPN14 as a YAP-binding protein that negatively regulates YAP activity by controlling its localization. Mechanistically, we find that the interaction of ectopic YAP with PTPN14 can be mediated by the respective WW and PPxY motifs. However, the PTPN14 PPxY motif and phosphatase activity appear to be dispensable for the negative regulation of endogenous YAP, likely suggesting more complex mechanisms of interaction and modulation. Finally, we demonstrate that PTPN14 downregulation can phenocopy YAP activation in mammary epithelial cells and synergize with YAP to induce oncogenic transformation. [ABSTRACT FROM AUTHOR]

Long chain alkylphenols are man-made compounds still present in industrial and agricultural processes. Their main use is domestic and they are widespread in household products, cleansers and cosmetics, leading to a global environmental and human contamination. These molecules are known to exert estrogen-like activities through binding to classical estrogen receptors. In vitro, they can also interact with the G-protein coupled estrogen receptor. Testicular germ cell tumor etiology and progression are proposed to be stimulated by lifelong estrogeno-mimetic exposure. We studied the transduction signaling pathways through which an alkyphenol mixture triggers testicular cancer cell proliferation in vitro and in vivo. Proliferation assays were monitored after exposure to a realistic mixture of 4-tert-octylphenol and 4-nonylphenol of either TCam-2 seminoma derived cells, NT2/D1 embryonal carcinoma cells or testis tumor in xenografted nude mice. Specific pharmacological inhibitors and gene-silencing strategies were used in TCam-2 cells in order to demonstrate that the alkylphenol mix triggers CREB-phosphorylation through a rapid, ERα36-PI3kinase non genomic pathway. Microarray analysis of the mixture target genes revealed that this pathway can modulate the expression of the DNA-methyltransferase-3 (Dnmt3) gene family which is involved in DNA methylation control. Our results highlight a key role for ERα36 in alkylphenol non genomic signaling in testicular germ cell tumors. Hence, ERα36-dependent control of the epigenetic status opens the way for the understanding of the link between endocrine disruptor exposure and the burden of hormone sensitive cancers. [ABSTRACT FROM AUTHOR]

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]