Min-Hyuk Yoo

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype and Tumorigenicity of Lung Carcinoma Cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

Targeting Thioredoxin Reductase 1 Reduction in Cancer Cells Inhibits Self-Sufficient Growth and DNA Replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1s role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

Selenoproteins that function in cancer prevention and promotion.

Of the many health benefits attributed to selenium, the one that has received the most attention is its role in cancer prevention. Selenium-containing proteins (selenoproteins) have been shown in recent years to have roles in cancer prevention. However, selenoproteins have diverse functions and their view as antioxidants is oversimplified. Some selenoproteins appear to have a split personality in having roles both in preventing and promoting cancer. The contrasting roles of one selenoprotein, thioredoxin reductase 1, in cancer are discussed in detail, but as also noted, at least one other selenoprotein may also have such a dual function. In addition, we discuss examples of inhibition of cancer development by selenoprotein deficiency in mouse models. These studies highlight the complex nature of selenium in relation to cancer.

Biosynthesis of Selenocysteine, the 21st Amino Acid in the Genetic Code, and a Novel Pathway for Cysteine Biosynthesis

The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA([Ser]Sec), is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA([Ser]Sec). Sec synthase (SecS) then uses O-phosphoseryl-tRNA([Ser]Sec) and the active donor of selenium, selenophosphate, to form Sec-tRNA([Ser]Sec). Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA([Ser]Sec) in the presence of SecS to form cysteine-(Cys-)tRNA([Ser]Sec). This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.

Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic 75Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 ± 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. Cancer Prev Res; 3(5); 630–9. ©2010 AACR.

Sep15, a thioredoxin-like selenoprotein, is involved in the unfolded protein response and differentially regulated by adaptive and acute ER stresses.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in activation of signaling pathways collectively known as the unfolded protein response (UPR). The UPR promotes adaptation of cells to ER stress by transient inhibition of protein translation and transcriptional up-regulation of genes encoding chaperones, oxidoreductases, and ER-associated degradation components. However, it may also trigger apoptosis in response to persistent ER stress. Recently, a novel selenocysteine-containing oxidoreductase, Sep15, has been reported to reside in the ER lumen. It has been proposed that this oxidoreductase may assist oxidative folding and structural maturation of N-glycosylated proteins targeted by UDP-glucose:glycoprotein glucosyltransferase, a chaperone implicated in quality control in the ER that forms a 1:1 complex with Sep15. To address the role of Sep15 in protein folding, we analyzed changes in Sep15 expression in murine fibroblast NIH3T3 cells in response to tunicamycin, brefeldin A (brefA), thapsigargin, and DTT that lead to accumulation of unfolded proteins within the ER. We show that expression of this protein is transcriptionally up-regulated in response to adaptive UPR caused by tunicamycin and brefA, whereas acute ER stress caused by DTT and thapsigargin leads to rapid and specific degradation of Sep15 by proteasomes. However, Sep15 deficiency did not result in detectable ER stress, consistent with the idea that Sep15 assists in the maturation of a restricted group of N-glycosylated proteins and/or that its function may be compensated by other mechanisms.

Thioredoxin reductase 1 protects against chemically induced hepatocarcinogenesis via control of cellular redox homeostasis

Thioredoxin reductase 1 (TR1) controls the redox state of protein thiols in mammalian cells and has been shown to have roles in both preventing and promoting cancer. To define the role of this selenoenzyme in hepatocellular carcinoma development, we examined tumor incidence in the liver of mice with tissue-specific knockout of mouse TR1 subjected to the liver carcinogen, diethylnitrosamine (DEN). TR1-deficient livers manifested ~90% tumor incidence compared with ~16% in control livers. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. On the other hand, we observed upregulation of another selenoenzyme, glutathione peroxidase 2 (GPx2), and components of the glutathione (GSH) system, including those that generate reduced GSH. Overall, this study shows that TR1 protects against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal.

Mouse Models Targeting Selenocysteine tRNA Expression for Elucidating the Role of Selenoproteins in Health and Development

Selenium (Se) deficiency has been known for many years to be associated with disease, impaired growth and a variety of other metabolic disorders in mammals. Only recently has the major role that Se-containing proteins, designated selenoproteins, play in many aspects of health and development begun to emerge. Se is incorporated into protein by way of the Se-containing amino acid, selenocysteine (Sec). The synthesis of selenoproteins is dependent on Sec tRNA for insertion of Sec, the 21st amino acid in the genetic code, into protein. We have taken advantage of this dependency to modulate the expression of Sec tRNA that in turn modulates the expression of selenoproteins by generating transgenic, conditional knockout, transgenic/standard knockout and transgenic/conditional knockout mouse models, all of which involve the Sec tRNA gene, to elucidate the intracellular roles of this protein class.

Deficiency in the 15 kDa Selenoprotein Inhibits Human Colon Cancer Cell Growth

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G0/G1 phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

Small hairpin RNA (shRNA) is a powerful tool for inhibiting gene expression. One limitation has been that this technique has been used primarily to target a single gene. This protocol expands upon previous methods by describing a knockdown vector that facilitates cloning of multiple shRNAs; this allows targeted knockdown of more than one gene or of a single gene that may otherwise be difficult to knockdown using a single shRNA. The targeted gene(s) can be readily re-expressed by transfecting knockdown cells with a knock-in vector, containing an shRNA-refractive cDNA that will express the protein-of-interest even in the presence of shRNAs. The constructed knockdown and knock-in vectors can be easily used concurrently to assess possible interrelationships between genes, the effects of gene loss on cell function and/or their restoration by replacing targeted genes one at a time. The entire knockdown or knock-in procedure can be completed in ∼3–4 months.