Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail.

Gigaxonin is an E3 ubiquitin ligase that plays a role in cytoskeletal stability. Its role in cancer is not yet clearly understood. Our previous studies of head and neck cancer had identified gigaxonin interacting with p16 for NFκB ubiquitination. To explore its role in cancer cell growth suppression, we analyzed normal and tumor DNA from cervical and head and neck cancers. There was a higher frequency of exon 8 SNP (c.1293 C>T, rs2608555) in the tumor (46% vs. 25% normal, P = 0.011) pointing to a relationship to cancer. Comparison of primary tumor with recurrence and metastasis did not reveal a statistical significance. Two cervical cancer cell lines, ME180 and HT3 harboring exon 8 SNP and showing T allele expression correlated with higher gigaxonin expression, reduced in vitro cell growth and enhanced cisplatin sensitivity in comparison with C allele expressing cancer cell lines. Loss of gigaxonin expression in ME180 cells through CRISPR-Cas9 or siRNA led to aggressive cancer cell growth including increased migration and Matrigel invasion. The in vitro cell growth phenotypes were reversed with re-expression of gigaxonin. Suppression of cell growth correlated with reduced Snail and increased e-cadherin expression. Mouse tail vein injection studies showed increased lung metastasis of cells with low gigaxonin expression and reduced metastasis with reexpression of gigaxonin. We have found an association between C allele expression and RNA instability and absence of multimeric protein formation. From our results, we conclude that gigaxonin expression is associated with suppression of epithelial-mesenchymal transition through inhibition of Snail. SIGNIFICANCE: Our results suggest that GAN gene exon 8 SNP T allele expression correlates with higher gigaxonin expression and suppression of aggressive cancer cell growth. There is downregulation of Snail and upregulation of e-cadherin through NFκB ubiquitination. We hypothesize that exon 8 T allele and gigaxonin expression could serve as diagnostic markers of suppression of aggressive growth of head and neck cancer.

Acidification of intracellular pH in MM tumor cells overcomes resistance to hypoxia-mediated apoptosis in vitro and in vivo.

Introduction: Multiple myeloma (MM) is an incurable cancer of malignant plasma cells that engraft in the bone marrow (BM). It is more than likely that the poorly investigated physical parameters of hypoxia and pH in the tumor microenvironment (TME) is critical for MM survival. Here, we explore the effects of a hypoxic environment on pH regulation and its role in MM survival. Methods: We used in vitro models of MM, in which the culturing medium was modified to specific pH and pO2 levels and then measured the effects on cell survival that was correlated with changes in intracellular (pHi) and extracellular pH (pHe). In a MM xenograft model, we used PET/CT to study hypoxia-mediated effects on tumor growth. Results: Hypoxia-mediated apoptosis of MM cells is correlated with acidic intracellular pHi (less than < 6.6) that is dependent on HIF activity. Using a polyamide HIF responsive element binding compound, a carbonic anhydrase inhibitor (acetazolamide), and an NHE-1 inhibitor (amiloride) acidified the pHi and lead to cell death. In contrast, treatment of cells with an alkalization agent, Na-lactate, rescued these cells by increasing the pHi (pH > 6.6). Finally, treatment of mice with acetazolamide decreased cell growth in the tumor nodules. Discussion: Targeting hypoxia and HIF have been proposed as an anti-tumor therapy but the clinical efficacy of such strategies are modest. We propose that targeting the pHi may be more effective at treating cancers within a hypoxic TME.

Can Targeting Hypoxia-Mediated Acidification of the Bone Marrow Microenvironment Kill Myeloma Tumor Cells?

Multiple myeloma (MM) is an incurable cancer arising from malignant plasma cells that engraft in the bone marrow (BM). The physiology of these cancer cells within the BM microenvironment (TME) plays a critical role in MM development. These processes may be similar to what has been observed in the TME of other (non-hematological) solid tumors. It has been long reported that within the BM, vascular endothelial growth factor (VEGF), increased angiogenesis and microvessel density, and activation of hypoxia-induced transcription factors (HIF) are correlated with MM progression but despite a great deal of effort and some modest preclinical success the overall clinical efficacy of using anti-angiogenic and hypoxia-targeting strategies, has been limited. This review will explore the hypothesis that the TME of MM engrafted in the BM is distinctly different from non-hematological-derived solid tumors calling into question how effective these strategies may be against MM. We further identify other hypoxia-mediated effectors, such as hypoxia-mediated acidification of the TME, oxygen-dependent metabolic changes, and the generation of reactive oxygen species (ROS), that may prove to be more effective targets against MM.

AcidoCEST MRI Evaluates the Bone Microenvironment in Multiple Myeloma.

PURPOSE: Multiple myeloma (MM) is an incurable disease of malignant plasma cells in the bone marrow (BM). Adaptive responses to hypoxia may be an essential element in MM progression and drug resistance. This metabolic adaptation involves a decrease in extracellular pH (pHe), and it depends on the upregulation of glucose transporters (GLUTs) that is common in hypoxia and in cancer cells. CEST MRI is an imaging technique that assesses pHe indirectly by the exchange rate of magnetic saturation transfer between labile protons on a solute and water. Thus, this study aimed to determine the feasibility of acidoCEST MRI for pHe measurement using an orthotopic mouse model of MM compared with GLUT1 immunofluorescence staining as a reference. PROCEDURES: Orthotopic BM engrafted MM xenografts were established in NSG/NOD mice using the human RPMI8226 myeloma cell line. AcidoCEST MRI was performed approximately 6 weeks after intravenous challenge, before and after intravenous administration of iopamidol. BM pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations. Samples were decalcified, sectioned, and immunostained for GLUT1 expression. Pearson's correlation was used to assess the relationship between pHe and [H3O+] versus GLUT1 expression. RESULTS: Ten mice underwent acidoCEST MRI followed by immunofluorescent histologic analysis. A strong negative correlation was seen between pHe versus GLUT1 expression (r = - 0.75, p < 0.001). After transformation of pH to [H3O+], a strong positive correlation between [H3O+] and GLUT1 expression was observed (r = 0.8, p < 0.001). CONCLUSIONS: AcidoCEST MRI can measure the extracellular pH of bone marrow affected by multiple myeloma. In this MM orthotopic mouse model, pHe measured by acidoCEST MRI showed strong correlations with the metabolic phenotype of BM tumor assessed by immunofluorescent histological assessment of GLUT1 overexpression.

A Novel Therapeutic Induces DEPTOR Degradation in Multiple Myeloma Cells with Resulting Tumor Cytotoxicity.

Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR-DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Restoration of the prolyl-hydroxylase domain protein-3 oxygen-sensing mechanism is responsible for regulation of HIF2α expression and induction of sensitivity of myeloma cells to hypoxia-mediated apoptosis.

Multiple myeloma (MM) is an incurable disease of malignant plasma B-cells that infiltrate the bone marrow (BM), resulting in bone destruction, anemia, renal impairment and infections. Physiologically, the BM microenvironment is hypoxic and this promotes MM progression and contributes to resistance to chemotherapy. Since aberrant hypoxic responses may result in the selection of more aggressive tumor phenotypes, we hypothesized that targeting the hypoxia-inducible factor (HIF) pathways will be an effective anti-MM therapeutic strategy. We demonstrated that MM cells are resistant to hypoxia-mediated apoptosis in vivo and in vitro, and that constitutive expression of HIF2α contributed to this resistance. Since epigenetic silencing of the prolyl-hydroxylase-domain-3 (PHD3) enzyme responsible for the O2-dependent regulation of HIF2α is frequently observed in MM tumors, we asked if PHD3 plays a role in regulating sensitivity to hypoxia. We found that restoring PHD3 expression using a lentivirus vector or overcoming PHD3 epigenetic silencing using a demethyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-dC), rescued O2-dependent regulation of HIF2α and restored sensitivity of MM cells to hypoxia-mediated apoptosis. This provides a rationale for targeting the PHD3-mediated regulation of the adaptive cellular hypoxic response in MM and suggests that targeting the O2-sensing pathway, alone or in combination with other anti-myeloma chemotherapeutics, may have clinical efficacy.

Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells.

DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase in TORC1 and TORC2 complexes. Overexpression of DEPTOR specifically occurs in a model of multiple myeloma. Its silencing in multiple myeloma cells is sufficient to induce cytotoxicity, suggesting that DEPTOR is a potential therapeutic target. mTORC1 paralysis protects multiple myeloma cells against DEPTOR silencing, implicating mTORC1 in the critical role of DEPTOR in multiple myeloma cell viability. Building on this foundation, we interrogated a small-molecule library for compounds that prevent DEPTOR binding to mTOR in a yeast-two-hybrid assay. One compound was identified that also prevented DEPTOR-mTOR binding in human myeloma cells, with subsequent activation of mTORC1 and mTORC2. In a surface plasmon resonance (SPR) assay, the compound bound to recombinant DEPTOR but not to mTOR. The drug also prevented binding of recombinant DEPTOR to mTOR in the SPR assay. Remarkably, although activating TORC1 and TORC2, the compound induced apoptosis and cell-cycle arrest in multiple myeloma cell lines and prevented outgrowth of human multiple myeloma cells in immunodeficient mice. In vitro cytotoxicity against multiple myeloma cell lines was directly correlated with DEPTOR protein expression and was mediated, in part, by the activation of TORC1 and induction of p21 expression. Additional cytotoxicity was seen against primary multiple myeloma cells, whereas normal hematopoietic colony formation was unaffected. These results further support DEPTOR as a viable therapeutic target in multiple myeloma and suggest an effective strategy of preventing binding of DEPTOR to mTOR. Cancer Res; 76(19); 5822-31. ©2016 AACR.

SGK Kinase Activity in Multiple Myeloma Cells Protects against ER Stress Apoptosis via a SEK-Dependent Mechanism.

UNLABELLED: To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib. IMPLICATIONS: Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.

A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity.

UNLABELLED: Multiple myeloma is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to multiple myeloma progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIF), and targeting HIFs can have anticancer effects in both solid and hematologic malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent, and this could be explained by the differential expression of the regulatory prolyl hydroxylase isoforms. The anti-multiple myeloma effects of a sequence-specific DNA-binding pyrrole-imidazole (Py-Im) polyamide (HIF-PA), which disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable with that of HIFs. Most of the multiple myeloma cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O2-dependent activation of HIFα using polyamides. IMPLICATIONS: Py-Im polyamides target and disrupt the adaptive hypoxic responses in multiple myeloma cells that may have clinical significance as a therapeutic strategy to treat myeloma engrafted in the bone marrow microenvironment.

MNK1-induced eIF-4E phosphorylation in myeloma cells: a pathway mediating IL-6-induced expansion and expression of genes involved in metabolic and proteotoxic responses.

Because multiple myeloma (MM) cells are at risk for endoplasmic reticulum (ER) stress, they require a carefully regulated mechanism to promote protein translation of selected transcripts when proliferation is stimulated. MAPK-interacting kinases (MNKs) may provide this mechanism by enhancing cap-dependent translation of a small number of critical transcripts. We, thus, tested whether MNKs played a role in MM responses to the myeloma growth factor interleukin-6 (IL-6). IL-6 activated MNK1 phosphorylation and induced phosphorylation of its substrate, eIF-4E, in MM lines and primary specimens. MNK paralysis, achieved pharmacologically or by shRNA, prevented MM expansion stimulated by IL-6. A phosphodefective eIF-4E mutant also prevented the IL-6 response, supporting the notion that MNK's role was via phosphorylation of eIF-4E. Both pharmacological MNK inhibition and expression of the phosphodefective eIF-4E mutant inhibited MM growth in mice. Although critical for IL-6-induced expansion, eIF-4E phosphorylation had no significant effect on global translation or Ig expression. Deep sequencing of ribosome-protected mRNAs revealed a repertoire of genes involved in metabolic processes and ER stress modulation whose translation was regulated by eIF-4E phosphorylation. These data indicate MM cells exploit the MNK/eIF-4E pathway for selective mRNA translation without enhancing global translation and risking ER stress.

DEPTOR is linked to a TORC1-p21 survival proliferation pathway in multiple myeloma cells.

We investigated the mechanism by which gene silencing of the mTOR inhibitor, DEPTOR, induces cytoreductive effects on multiple myeloma (MM) cells. DEPTOR knockdown resulted in anti-MM effects in several MM cell lines. Using an inducible shRNA to silence DEPTOR, 8226 MM cells underwent TORC1 activation, downregulation of AKT/SGK activity, apoptosis, cell cycle arrest and senescence. These latter cytotoxic effects were prevented by TORC1 paralysis (Raptor knockdown) but not by over-expression of AKT activity. In addition, DEPTOR knockdown-induced MM death was not associated with activation of the unfolded protein response, suggesting that enhanced ER stress did not play a role. In contrast, DEPTOR knockdown in 8226 cells induced p21 expression, independent of p53, and p21 knockdown prevented all of the cytotoxic effects following DEPTOR silencing. DEPTOR silencing resulted in p21 upregulation in additional MM cell lines. Furthermore, DEPTOR silencing in a murine xenograft model resulted in anti-MM effects associated with p21 upregulation. DEPTOR knockdown also resulted in a decreased expression of p21-targeting miRNAs and transfection of miRNA mimics prevented p21 upregulation and apoptosis following DEPTOR silencing. Use of a shRNA-resistant DEPTOR construct ruled out off-target effects of the shRNA. These results indicate that DEPTOR regulates growth and survival of MM cells via a TORC1/p21 pathway and suggest an involvement of p21-targeted miRNAs.

Metabolomics identifies pyrimidine starvation as the mechanism of 5-aminoimidazole-4-carboxamide-1-ß-riboside-induced apoptosis in multiple myeloma cells.

To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl-α-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce multiple myeloma cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in multiple myeloma cells.

Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis.

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

Dietary ß-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection.

The effect of ß-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with ß-glucan (MacroGard®) for 14 days with a daily ß-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 108 bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of ß-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a ß-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the ß-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the ß-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.

PAK1 kinase promotes cell motility and invasiveness through CRK-II serine phosphorylation in non-small cell lung cancer cells.

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.

The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance.

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with ß-glucan supplements.

The objective of the present study was to determine the action of ß-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. ß-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1ß, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with ß-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed ß-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1ß and il6 in infected fish fed ß-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected ß-glucan group. In conclusion, a diet supplemented with ß-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.

Molecular cloning and expression of two ß-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after ß-glucan feeding.

In this study, we described the partial structure, mRNA tissue distribution and regulation of two carp mucin and two ß-defensin genes. This is the first description of these genes in fish. The genes might provide relevant tools to monitor feed-related improvements of fish health under aquaculture conditions. Carp mucin 2 and mucin 5B genes show a high similarity to their mammalian and avian counterparts. The carp ß-defensin 1 and ß-defensin 2 genes cluster together well with their piscine family members. The influence of a ß-glucan immunomodulant on the expression of these genes in mucosal tissues could be confirmed for the first time. Muc5B expression was significantly increased in the skin. For Muc2 no significant up- or down-regulation could be observed. Significantly higher expression levels of ß-defensin 2 in gills and both ß-defensin genes in skin were found. Thus, the mucosal system can be influenced by the addition of ß-glucans to the food.

IL-6-induced enhancement of c-Myc translation in multiple myeloma cells: critical role of cytoplasmic localization of the rna-binding protein hnRNP A1.

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5'-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.