Melissa L. T. Teoh

Overexpression of extracellular superoxide dismutase attenuates heparanase expression and inhibits breast carcinoma cell growth and invasion

Increased expression of heparanase stimulates the progression of various human cancers, including breast cancer. Therefore, a deeper understanding of the mechanisms involved in regulating heparanase is critical in developing effective treatments for heparanase-overexpressing cancers. In this study, we investigated the potential use of extracellular superoxide dismutase (EcSOD) to enhance the inhibitory effects of heparin/low molecular weight heparin (LMWH) in breast cancer cells. EcSOD binds to cell surfaces and the extracellular matrix through heparin-binding domain (HBD). Deleting this HBD rendered the protein a more potent inhibitor of breast cancer growth, survival, and invasion. Among the treatment combinations examined, EcSODDeltaHBD plus LMWH provided the best tumor suppressive effects in inhibiting breast cancer growth and invasion in vitro. We have further shown that overexpression of EcSOD decreased accumulation of vascular endothelial growth factor in the culture medium and increased the level of intact cell surface-associated heparan sulfate, thus implicating inhibition of heparanase expression as a potential mechanism. Overexpression of EcSOD inhibited steady-state heparanase mRNA levels by >50% as determined by quantitative reverse transcription-PCR. Moreover, heparanase promoter activation was suppressed by EcSOD as indicated by a luciferase reporter assay. These findings reveal a previously unrecognized molecular pathway showing that regulation of heparanase transcription can be mediated by oxidative stress. Our study implies that overexpression of EcSOD is a promising strategy to enhance the efficacy of heparin/LMWH by inhibiting heparanase as a novel treatment for breast cancer.

Modulation of Reactive Oxygen Species in Pancreatic Cancer

Purpose: The aim of the present study was to compare the effects of the three different forms of the antioxidant enzyme superoxide dismutase [i.e., manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and extracellular superoxide dismutase (EcSOD)] on the malignant phenotype of human pancreatic cancer. Experimental Design: Human pancreatic cancer cell lines were infected with adenoviral vectors containing the cDNAs for three different forms of the antioxidant enzyme SOD. Intratumoral injections of the adenoviral vectors were used in nude mice with human tumor xenografts. Results: Increases in immunoreactive protein and enzymatic activity were seen after infections with the AdMnSOD, AdCuZnSOD, or AdEcSOD constructs. Increased SOD activity decreased superoxide levels and increased hydrogen peroxide levels. Increasing SOD levels correlated with increased doubling time. Cell growth and plating efficiency decreased with increasing amounts of the adenoviral constructs, with the AdCuZnSOD vector having the greatest effect in decreasing in vitro tumor growth. In contrast, inhibiting endogenous SOD with small interfering RNA increased superoxide levels and promoted tumor growth. Of the three SODs, tumors grew the slowest and survival was increased the greatest in nude mice injected with the AdEcSOD construct. Conclusions: Scavenging plasma membrane–generated superoxide may prove beneficial for suppression of pancreatic cancer growth.

Aberrant promoter CpG methylation is a mechanism for impaired PHD3 expression in a diverse set of malignant cells.

Background The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. Methodology/Principal Findings Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3. Conclusions/Significance We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Leporipoxvirus Cu,Zn-Superoxide Dismutase (SOD) Homologs Are Catalytically Inert Decoy Proteins That Bind Copper Chaperone for SOD

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.

REGULATION OF PANCREATIC CANCER GROWTH BY SUPEROXIDE

K‐ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K‐ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K‐ras oncogene correlates with increased non‐mitochondrial‐generated superoxide (O  2.− ), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er‐Kras+ (H6c7 cells expressing K‐ras oncogene), and H6c7eR‐KrasT (tumorigenic H6c7 cells expressing K‐ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K‐ras. Western blots and activity assays for the antioxidant enzymes that detoxify O  2.− were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non‐mitochondrial source of the increased levels of O  2.− , Western analysis demonstrated the absence of NADPH oxidase‐2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K‐ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K‐ras oncogene, overexpression of superoxide dismutases that detoxify non‐mitochondrial sources of O  2.− , and treatment with the small molecule O  2.− scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O  2.− produced by NOX2 in pancreatic cancer cells with K‐ras, may regulate pancreatic cancer cell growth.

Human chondrosarcoma cells acquire an epithelial-like gene expression pattern via an epigenetic switch: Evidence for mesenchymal-epithelial transition during sarcomagenesis

Chondrocytes are mesenchymally derived cells that reportedly acquire some epithelial characteristics; however, whether this is a progression through a mesenchymal to epithelial transition (MET) during chondrosarcoma development is still a matter of investigation. We observed that chondrosarcoma cells acquired the expression of four epithelial markers, E-cadherin,desmocollin 3, maspin, and 14-3-3σ, all of which are governed epigenetically through cytosine methylation. Indeed, loss of cytosine methylation was tightly associated with acquired expression of both maspin and 14-3-3σ in chondrosarcomas. In contrast, chondrocyte cells were negative for maspin and 14-3-3σ and displayed nearly complete DNA methylation. Robust activation of these genes was also observed in chondrocyte cells following 5-aza-dC treatment. We also examined the transcription factor snail which has been reported to be an important mediator of epithelial to mesenchymal transitions (EMTs). In chondrosarcoma cells snail is downregulated suggesting a role for loss of snail expression in lineage maintenance. Taken together, these results document an epigenetic switch associated with an MET-like phenomenon that accompanies chondrosarcoma progression.

Tumorigenic Poxviruses Up-Regulate Intracellular Superoxide To Inhibit Apoptosis and Promote Cell Proliferation

ABSTRACT Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RΔ virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RΔ virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.

Transgenic biosynthesis of trypanothione protects Escherichia coli from radiation-induced toxicity.

Abstract Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H2DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by 137Cs &ggr; radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent.

Abstract 2285: Epigenetic silencing of SOD3 contributes to the malignant phenotype in human lung cancer

Extracellular SOD (EcSOD), encoded by the SOD3 gene, scavenges superoxide in the extracellular space. EcSOD is highly expressed in the lung where it comprises the majority of SOD activity in airway epithelial and vascular cells. The role of EcSOD in lung cancer, if any, has not yet been thoroughly studied. It has been reported, however, that EcSOD expression is lost in the lung cancer cell line A549 and that the transcription factors SP1/SP3 that regulate expression in MRC-5 cells could neither bind the promoter nor stimulate EcSOD expression in A549 cells. We hypothesized that EcSOD loss in lung cancer could be due to epigenetic events. To test this hypothesis we analyzed the epigenotype of the SOD3 promoter in three normal cell types (airway epithelial, MRC-5, HMEC) and two EcSOD negative lung cancer cell lines (A549, H1650) as well as in normal lung versus five human lung tumors. We also re-expressed EcSOD in the A549 cell line to assess possible changes in malignant phenotype. We now report that EcSOD expression is highly correlated with SOD3 promoter methylation. In the normal airway epithelial and MRC-5 lung cells that express EcSOD, the promoter has an overall cytosine methylation of 86% methylation at the 18 CpG sites examined. Treatment of cells with 5-aza-dC induced SOD3 mRNA, indicating that DNA methylation was causal, at least in part, to the gene silencing. Moreover, A549 and H1650 cells displayed a ∼25-fold reduction in chromatin accessibility of the SOD3 promoter compared to MRC-5 cells. In the five lung tumors, EcSOD mRNA expression was significantly decreased and the SOD3 promoter showed a significantly more methylated state in the tumors compared to normal lung tissue. Re-expression of EcSOD attenuated the malignant phenotype of A549 cells by increasing doubling time by 25 hours, decreasing matrigel invasion and clonogenic capacity by 65% and 70% respectively. Taken together, our findings indicate that loss of EcSOD expression in human lung cancer is highly correlated with aberrant SOD3 promoter methylation and a repressive chromatin structure, and that re-acquisition of EcSOD attenuates the malignant phenotype of lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2285.

Abstract 3373: Overexpression of EcSOD-ΔHBD suppresses breast cancer metastasis

A critical event in the process of cancer invasion and metastasis is the remodeling of the extracellular matrix (ECM). Heparanase participates in this event by degrading the heparan sulfate (HS) component of the cell surface and the ECM. The expression level of heparanase correlates with an aggressive phenotype and greater metastasis potential in most cancers. Therefore, a variety of heparanase inhibitors, including low molecular weight heparin (LMWH) have been developed as anticancer agents. An overlooked yet interesting effect of LMWH administration is an increase in the circulating level of extracellular superoxide dismutase (EcSOD), which may play a role in its anti-cancer function. We have previously shown that overexpression of EcSOD enhanced the inhibitory effect of LMWH on breast carcinoma growth, clonogenic survival, and invasion in vitro, partly by attenuating heparanase expression and preventing HS degradation. To further understand the role of EcSOD in breast cancer progression in vivo, we performed an experimental lung metastasis study in which MDA-MB231.luc cells were introduced intravenously into athymic nude mice and colonization of the cells to the lungs was monitored by bioluminescent imaging. Overexpression of EcSOD via intramuscular injection of adenovirus vector resulted in a significant reduction in lung colonization compared to the empty vector (AdEmpty) infected group by more than two fold. Strikingly, a ten fold suppression of lung metastasis was observed when a truncated form of EcSOD that has a deletion in its heparin binding domain, EcSODΔHBD was introduced. Furthermore, EcSOD also significantly prolonged the survival of the animals from 58 days in AdEmpty group to 78 days in the AdEcSOD group and 99 days in the AdEcSOD⊗HBD group. In addition to studying the lung colonization, we also determined he effect of EcSOD on a spontaneous metastasis using a yngeneic model whereby a highly aggressive mouse mammary carcinoma cell line, 4T1.luc was injected into the mammary fat pad of immuno-competent BALB/c mice. Bioluminescent imaging analysis indicated that when all animals in the AdEmpty group showed secondary metastasis, only 71% and 63% of mice infected with AdEcSOD and AdEcSODΔHBD, respectively, developed secondary metastasis. Moreover, both EcSOD and EcSODΔHBD prolonged the survival of the animals from 44 days in the AdEmpty group to 69 days in the AdEcSOD group to 77 days in the AdEcSODΔHBD group. These results clearly indicate that EcSOD plays an important role in suppressing breast cancer metastasis and that the EcSODΔHBD that remains in the circulation system is more efficient than the full length protein. Most significantly, this is the first study demonstrating the biological function of the truncated EcSOD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3373.