Ras-transformed NIH 3T3 Research Articles

Preparation and antitumor activities of β-azatyrosinamides

A large number of human prostate cancer cases has been proven to be genetically associated with ras-mutation. A series of α-azatyrosinamides prepared in this laboratory demonstrated selective cytotoxicity against rasmutated NIH3T3 cells while with little toxicity on wild type NIH3T3 cells. The compounds also proved to be active in inhibiting human prostate cancer cell lines. Using α-azatyrosinamides as the leads, we prepared another series of novel β-azatyrosinamides for the purpose of treating human prostate cancer. Preparation of the β-azatyrosinamides starts from 5-benzyloxypyridin-2-ylaldehyde (1), which upon reaction with malonic acid and ammonium acetate afforded 5-benzyloxy-β-azatyrosine (2). This intermediate was allowed to react with benzyloxycarbonic anhydride followed by coupling with a variety of amines to form the desired β-azatyrosinamides 4-15. The compounds exhibited an inhibitory effect on the growth of ras-mutated NIH3T3 cells with IC50 ranged between 0.13 ± 0.01 mM and 3.16 ± 1.45 mM, which were with activities 2-57 fold higher than that of azatyrosine, but were much less active than their α-amino acid analogues. The selective toxicity, in terms of the ratio of IC50 against wild type NIH3T3 to that against ras-transformed NIH3T3 cell lines, is at the range of 0.7-11.9. The IC50's of β-azatyrosinamides 4-15 on PC-3 human prostate cancer cell line ranged between 0.15 ± 0.02 mM and 13.05 ± 9.41 mM, which were 0.4-33 fold lower than that of azatyrosine.

Antimetastatic effect of epigenetic drugs, hydralazine and valproic acid, in Ras-transformed NIH 3T3 cells.

IntroductionMetastasis involves the accumulation of genetic and epigenetic alterations leading to activation of prometastatic genes and inactivation of antimetastatic genes. Among epigenetic alterations, DNA hypermethylation and histone hypoacetylation are the focus of intense translational research because their pharmacological inhibition has been shown to produce antineoplastic activity in a variety of experimental models.AimsThis study aimed to evaluate the antimetastatic effect of the DNA-methylation inhibitor, hydralazine, and the histone deacetylase inhibitor, valproic acid.MethodsNIH 3T3-Ras murine cells were treated with hydralazine and valproic acid to evaluate their effects upon cell proliferation, cell motility, chemotaxis, gelatinase activity, and gene expression. Lung metastases were developed by intravenous injection of NIH 3T3-Ras cells in BALB/c nu/nu mice and then treated with the drug combination.ResultsTreatment induced a growth-inhibitory effect on NIH 3T3-Ras cells, showed a trend toward increased gelatinase activity of MMP2 and MMP9, and inhibited chemotaxis and cell motility. The combination led to a strong antimetastatic effect in lungs of nude mice.ConclusionHydralazine and valproic acid, two repositioned drugs as epigenetic agents, exhibit antimetastatic effects in vitro and in vivo and hold potential for cancer treatment.

T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

Recurrence and metastasis are major challenges in the management of hepatocellular carcinoma (HCC) patients after resection. To identify a metastasis-associated gene signature, we performed comparative gene expression analysis with recurrent HCC tissues from HCC patients who underwent partial or total hepatectomy and from non-metastatic primary HCC tissues. From this, we were able to identify genes associated with HCC recurrence. TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed genes in patients with recurrent HCC who had undergone total hepatectomy. The significant overexpression of TCIRG1 was confirmed using the Liver Hepatocellular Carcinoma dataset from The Cancer Genome Atlas. High expression of TCIRG1 was significantly associated with poor 5-year disease-free and recurrence-free survival of HCC patients. TCIRG1 knockdown suppressed tumor cell growth and proliferation in HCC cell lines; caused a significant increase in the proportion of cells in the G1/S phase of cell cycle; induced cell death; suppressed the metastatic potential of HCC cells by selectively regulating the epithelial–mesenchymal transition (EMT) regulatory proteins E-cadherin, N-cadherin, Fibronectin, Snail and Slug; and significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. These findings suggest that TCIRG1 functions as a metastatic enhancer by modulating growth, death and EMT in HCC cells. TCIRG1 could be a therapeutic target for the treatment of liver malignancy and metastasis.

Abstract 3829: TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis

Abstract Recurrence and metastasis are a major challenge in the management of hepatocellular carcinoma (HCC) patients after HCC resection. In order to identify metastatic molecular signature, we performed comparative gene expression profiling analysis with recurred HCC tissues from HCC patients who underwent partial or total hepatectomy and non-metastatic primary HCC. From this, we were able to recapitulate molecular signatures associate with HCC recurrence, and found that TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed gene elements in recurred HCC patients with total hepatectomy. We then observed that TCIRG1 is significantly overexpressed in TCGA-LIHC (Liver Hepatocellular Carcinoma Dataset of The Cancer Genome Atlas), and high expression of TCIRG1 was significantly associated with poor prognosis of HCC patients in 5-year disease-free. TCIRG1 knockdown suppressed tumor cell growth and proliferation in Huh7 and SNU475, liver cancer cell lines. From flow cytometric analysis for cell cycle analysis and cell death, we observed that TCIRG1 knockdown caused a significant increase of G1/S phase, and also induced programmed cell death processing in liver cancer cells. TCIRG1 knockdown also reduced the metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins, E-cadherin, N-cadherin, Vimentin, Fibronectin, Snail and Slug, in liver cancer cells. In addition, sustained expression of TCIRG1 in liver cancer cell line reduced tumor growth rate in mouse xenograft model. Moreover, targeted-disruption of TCIRG1 significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. In conclusion, our findings suggest that TCIRG1 functions as metastatic enhancing gene by modulating cellular growth, death, and EMT in liver cancer cells providing TCIRG1 as a therapeutic target for the treatment of liver malignancy and metastasis. Citation Format: Hee Doo Yang, Jung Woo Eun, Qingyu Shen, Hyung Seok Kim, Sang Yean Kim, Suk Woo Nam. TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2017-3829

Post-Transcriptional Regulation of Connexin43 in H-Ras-Transformed Cells

Connexin43 (Cx43) expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3′ and 5′Untranslated regions (UTRs) and found an opposing effect; a 5′UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3Ras), while the 3′UTR is active only in normal NIH-3T3Neo cells and completely silenced in NIH-3T3Ras cells. Most importantly, we identified a previously unknown regulatory element within the 3′UTR, named S1516, which accounts for this 3′UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3Neo compared to NIH-3T3Ras cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2

Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha- ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G 1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy—likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.

Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis

In cancer cells, glucose is often converted into lactic acid, which is known as the 'Warburg effect'. The reason that cancer cells have a higher rate of aerobic glycolysis, but not oxidative phosphorylation, remains largely unclear. Herein, we proposed an epigenetic mechanism of the Warburg effect. Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonize glycolysis was downregulated through NF-kappaB pathway in Ras-transformed NIH3T3 cells. Restoration of FBP1 expression suppressed anchorage-independent growth, indicating the relevance of FBP1 downregulation in carcinogenesis. Indeed, FBP1 was downregulated in gastric carcinomas (P<0.01, n=22) and gastric cancer cell lines (57%, 4/7). Restoration of FBP1 expression reduced growth and glycolysis in gastric cancer cells. Moreover, FBP1 downregulation was reversed by pharmacological demethylation. Its promoter was hypermethylated in gastric cancer cell lines (57%, 4/7) and gastric carcinomas (33%, 33/101). Inhibition of NF-kappaB restored FBP1 expression, partially through demethylation of FBP1 promoter. Notably, Cox regression analysis revealed FBP1 promoter methylation as an independent prognosis predicator for gastric cancer (hazard ratio: 3.60, P=0.010). In summary, we found that NF-kappaB functions downstream of Ras to promote epigenetic downregulation of FBP1. Promoter methylation of FBP1 can be used as a new biomarker for prognosis prediction of gastric cancer. Such an important epigenetic link between glycolysis and carcinogenesis partly explains the Warburg effect.

Overexpression of Plk3 causes Morphological Change and Cell Growth Suppression in Ras Pathway-activated Cells

To unravel the growth inhibition mechanism of Polo-like kinase 3 (Plk3), the effect of overexpression of Plk3 was examined in 293T cells. Cell rounding, changes in actin organization and cellular detachment were induced by Plk3 transfection in a kinase activity-dependent manner. Although apoptosis was not observed, Plk3 overexpression suppressed cellular growth in a long-term colony-forming assay. Because both Plk3 and Ras affect F-actin organization, the effect of co-transfection of Plk3 and Ras was evaluated. Adhesion was synergistically lost by co-transfection of these two genes, compared with transfection of Plk3 alone. Furthermore, overexpression of Plk3 caused long-term growth suppression in Ras-transformed NIH3T3. Collectively, Plk3 activation might cause cytoskeleton re-organization and result in growth suppression more pronouncedly in Ras pathway-activated cells.

Activated Ras/MEK Inhibits the Antiviral Response of Alpha Interferon by Reducing STAT2 Levels

The ability of interferon (IFN) to induce the expression of antiviral genes, and therefore suppress viral infection, is dependent on the activity of cellular suppressors. The Ras/MEK pathway is one of these cellular suppressors, since the activation of Ras/MEK permits viral replication in the presence of alpha IFN (IFN-alpha). Here, we have investigated the mechanism by which activated Ras/MEK inhibits the IFN-alpha response. We found that the induction of antiviral proteins in response to IFN-alpha was impaired in Ras-transformed NIH 3T3 (RasV12) cells. The inhibition of the Ras/MEK pathway restored the IFN-mediated induction of antiviral genes, indicating that activated Ras interrupts the IFN pathway upstream of antiviral gene transcription. Indeed, the IFN-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 was inhibited in RasV12 cells compared to that of vector control cells. In addition, we found that the total amount of STAT2 was reduced in RasV12 cells. To determine if the impaired IFN-alpha response can be rescued by restoring the overall level of STAT2, we overexpressed STAT2 in RasV12 cells. The IFN-alpha-induced phosphorylation of STAT1 and STAT2, as well as the expression of antiviral protein, were restored, and IFN-induced antiviral protection was partially restored. Moreover, we demonstrated that the downregulation of STAT2 levels by Ras/MEK was mediated at the transcriptional level. Thus, the activation of the Ras/MEK pathway reduces the amount of STAT2 available for propagating the IFN signal, resulting in the impairment of the IFN-alpha-induced antiviral response.

Induction of senescence by progesterone receptor-B activation in response to cAMP in ovarian cancer cells

Objective Progesterone receptor (PR) expression is a favorable prognostic marker in ovarian cancer. We previously demonstrated that the induction of PR-B by treatment with cAMP was associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH 3T3 cells. In this study, we examined the effect of cAMP treatment on cell growth in Ras-transformed NIH3T3 cells and ovarian cancer cells. Methods 1) The levels of PR-B and cell cycle associated proteins (p21, p27 and Rb) following treatment with cAMP in the Ras-transformed NIH3T3 cells (K12V) and ovarian cancer cell lines (SKOV cells) were investigated by Western blots. 2) The effects of PR overexpression following treatment with cAMP or after infection of an adenovirus expressing PR-B on cell growth and tumorigenicity in a soft agar culture were examined. Results 1) Treatment with cAMP increased PR-B and p27 levels in K12V cells and inhibited cell growth by inducing premature senescence. Induction of senescence was specific to the transformed cells. 2) In SKOV cells, treatment with cAMP induced PR-B, p27 and p21 expression, reduced the level of phosphorylated Rb, caused accumulation of cells in the G0/G1 fraction of the cell cycle, and induced senescence. 3) Both anchorage-dependent and -independent SKOV cell growths were inhibited by cAMP treatment. 4) Induction of both the expression and transcriptional activity of PR-B is critical for the induction of senescence and suppression of tumorigenicity. Conclusion Treatment of cAMP, through activation of PR-B, induced senescence and suppressed tumorigenicity in ovarian cancer cells.

Difference gel electrophoresis analysis of Ras‐transformed fibroblast cell‐derived exosomes

Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras-transformed NIH3T3 cells. The vesicular nature and size (30-100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2-D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras-transformed NIH3T3 cells. LC-MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen alpha-1 (VI), 14-3-3 isoforms, guanine nucleotide-binding proteins (G proteins), the eukaryotic translation initiation factors elF-3 gamma and elF-5A accumulated (>2-fold) in exosomes upon Ras-induced oncogenic transformation. Significantly, the 10.4-fold increase in v-Ha-Ras p21 protein in exosomes derived from Ras-transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.

Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs, via tyrosine phosphorylation at specific residues. We recently reported that FAK Tyr-407 phosphorylation negatively regulates the enzymatic and biological activities of FAK, unlike phosphorylation of other tyrosine residues. In this study, we further investigated the effect of FAK Tyr-407 phosphorylation on cell transformation. We found that FAK Tyr-407 phosphorylation was lower in H- Ras transformed NIH3T3 and K- Ras transformed rat-2 fibroblasts than in the respective untransformed control cells. Consistently, FAK Tyr-407 phosphorylation was decreased in parallel with cell transformation in H- Ras-inducible NIH3T3 cells and increased during trichostatin A-induced detransformation of both K- Ras transformed rat-2 fibroblasts and H- Ras transformed NIH3T3 cells. In addition, overexpression of a phosphorylation-mimicking FAK Tyr-407 mutant inhibited morphological transformation of H- Ras-inducible NIH3T3 cells and inhibited invasion activity and anchorage-independent growth of H- Ras-transformed NIH3T3 cells. Taken together, these data strongly suggest that FAK Tyr-407 phosphorylation negatively regulates transformation of fibroblasts.

Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels

The 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down-regulation of AMFR expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or perinuclear ER markers and only partially overlaps with the translocon component Sec61alpha. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain. Elevation of free cytosolic Ca(2+) levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy, disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced release of ER Ca(2+) stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptor (IP3R) colocalises to 3F3A-labelled mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 cells have increased basal cytosolic Ca(2+) levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our data indicate that regulation of the ER-mitochondria association by free cytosolic Ca(2+) is a characteristic of smooth ER domains and that multiple mechanisms regulate the interaction between these organelles.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Repression of BCL2 by the Tumor Suppressor Activity of the Lysyl Oxidase Propeptide Inhibits Transformed Phenotype of Lung and Pancreatic Cancer Cells

The gene encoding lysyl oxidase (LOX) was identified as the ras recision gene (rrg), with the ability to revert Ras-mediated transformation of NIH 3T3 fibroblasts. Mutations in RAS genes have been found in approximately 25% of lung cancers and in 85% of pancreatic cancers. In microarray analysis, these cancers were found to display reduced LOX gene expression. Thus, the ability of the LOX gene to repress the transformed phenotype of these cancer cells was tested. LOX is synthesized as a 50-kDa secreted precursor Pro-LOX that is processed to the 32-kDa active enzyme (LOX) and to an 18-kDa propeptide (LOX-PP). Recently, we mapped the rrg activity of Pro-LOX to the LOX-PP in Ras-transformed NIH 3T3 cells. Ectopic Pro-LOX and LOX-PP expression in H1299 lung cancer cells inhibited growth in soft agar and invasive colony formation in Matrigel and reduced activation of extracellular signal-regulated kinase (ERK) and Akt, with LOX-PP showing substantially higher activity. Similarly, LOX-PP expression in PANC-1 pancreatic cancer cells effectively reduced ERK and Akt activity and inhibited growth in soft agar and ability of these cells to migrate. Nuclear Factor-kappaB (NF-kappaB) and its target gene BCL2, which are overexpressed in 70% to 75% of pancreatic cancers, have recently been implicated in invasive phenotype. LOX-PP substantially reduced NF-kappaB and Bcl-2 levels. Reintroduction of Bcl-2 into PANC-1 or H1299 cells expressing LOX-PP restored the transformed phenotype, suggesting that Bcl-2 is an essential target. Thus, LOX-PP potently inhibits invasive phenotype of lung and pancreatic cancer cells, suggesting potential therapeutic applications in treatment of these cancers.

Analysis of Ras‐induced oncogenic transformation of NIH‐3T3 cells using differential‐display 2‐DE proteomics

Ras proteins control at least three crucial signalling networks responsible for several cellular processes including anchorage independence, survival, and proliferation. Point mutations in one of the three ras genes are frequent in human tumours. In these tumours, Ras oncoproteins contribute significantly to the malignant phenotype, including deregulation of tumour-cell growth, apoptosis and invasiveness, and the ability to induce angiogenesis. Although significant strides have been made in understanding Ras biology, the collaborative actions of Ras effectors are still poorly understood. Here, we describe a proteomics approach to study global changes in protein expression in Ras-transformed NIH3T3 cells. We exploited 2-D difference gel electrophoresis (DIGE) for pre-separation fluorescent protein labelling with three separate dyes to reduce gel-to-gel variability, to increase sensitivity and dynamic range of protein detection, and to enhance quantification of dysregulated proteins. Proteins dysregulated (> 1.5-fold) by oncogenic Ras transformation reported to be implicated in Ras-regulated pathways include S-methyl-5-thioadenosine phosphorylase, stress-induced-phosphoprotein 1, galectin-1, annexin A7 (synexin), 60S acidic ribosomal protein P0, serine/threonine protein phosphatase type 1 (PP1alpha) and prohibitin. Significantly, we report for the first time the expression of the newly discovered cytokine IL-25 (or IL-17E) in mouse embryonic fibroblast cells and its down-regulation (2.1-fold) upon Ras-induced oncogenic transformation.

Ras에 의해 암화된 세포에서 dynamin-2의 발현 촉진

Dynamin은 여러 종류의 endocytosis 과정에서 최종적으로 endocytic vesicle을 membrane으로부터 분리하는데 중요한 역할을 하는 단백질이다. 이전의 보고에 의하면 dynamin-2는 Ras에 의해 암화된 세포에서 Ras signal의 신호 전달 단백질인 Grb2의 SH3 domain과 결합한다고 알려져 있다. 하지만 정상적인 세포 (NIH3T3)에 비해 Ras에 의해 암화된 세포 (NIH3T3(Ras))에서 이들 단백질의 발현이 높아지는지에 대해서는 아직 알려진 바가 없다. 본 연구에서는 먼저 NIH3T3 세포와 NIH3T3(Ras) 세포에서 dynamin-2와 Grb2의 단백질 발현을 보았는데, dynamin-2의 경우 NIH3T3 세포에 비해 NIH3T3(Ras) 세포에서 그 발현이 현저히 증가함을 볼 수 있었지만 Grb2의 경우 두 세포에서 발현의 차이를 관찰할 수 없었다. Competitive PCR을 이용하여 mRNA의발현정도를 확인하였을 때, 단백질 발현 정도와 마찬가지로 dynamin-2의 경우 NIH3T3(Ras) 세포에서 약 100배의 증가를 확인하였지만 Grb2의 경우 차이를 볼 수 없었다. Dynamin-2의 promoter 활성을 NIH3T3(Ras) 세포에서 관찰한 결과 start codon으로부터 300 bp에서 200 bp upstream에 dynamin-2의 promoter 활성을 조절하는 부위가 존재함을 확인할 수 있었다. Dynamin plays a key role in the scission event common to various types of endocytosis. It has been previously reported that the SH3 domain-mediated association of Grb2 with dynamin-2 was dominantly found in Ras transformed cells. However, whether this association results from the increased expression of dynamin-2 and Grb2 in Ras transformed cells or not is still unknown. So in this study we first analyzed the expression levels of dynamin-2 and Grb2 and found that the expression of dynamin-2 protein was dramatically increased in Ras-transformed NIH3T3 (NIH3T3(Ras)) cells. Furthermore competitive PCR data revealed that the mRNA transcripts for dynamin-2 were increased about 100-fold in NIH3T3(Ras) compared to those of NIH3T3 cells. However, the protein level and mRNA transcript of Grb2 were not changed in these two cells. We also examined promoter activity of dynamin-2 in NIH3T3(Ras) cells and suggest the existence of Ras-responsive sequence in promoter region -300 to -200.

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.