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The 15kDa selenoprotein and thioredoxin reductase 1 promote colon cancer by different pathways.

Tsuji PA, Carlson BA, Yoo MH, Naranjo-Suarez S, Xu XM, He Y, Asaki E, Seifried HE, Reinhold WC, Davis CD, Gladyshev VN, Hatfield DL - PLoS ONE (2015)

Bottom Line: We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells.These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells.Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Towson University, Towson, Maryland, United States of America.

ABSTRACT
Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

No MeSH data available.


Related in: MedlinePlus

Validation of gene expression for genes significantly changed in shSep15 cells.Expression of (A) Ifi44 mRNA (upper panel) and protein (lower panel); (B) Irf-7 mRNA; (C) Usp18 mRNA; (D) Gbp-6 mRNA; (E) Ifnγ mRNA; (F) Gbp-1 mRNA (upper panel) and protein (lower panel); (G) Stat-1 mRNA (upper panel) and protein (lower panel); (H), Casp6 mRNA; (I) Casp12 mRNA; and (J) Afp mRNA. Expression of mRNA in control, shSep15, shTR1, and shTR1/Sep15 cells was determined by real-time RT-PCR, and expressed relative to Gapdh. Columns, mean (n = 3–6); bars, SE; (*P<0.05, **P<0.01, ***P<0.001). Protein expression was determined by Western blotting, and expressed relative to β-actin or α-tubulin, as indicated.
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pone.0124487.g004: Validation of gene expression for genes significantly changed in shSep15 cells.Expression of (A) Ifi44 mRNA (upper panel) and protein (lower panel); (B) Irf-7 mRNA; (C) Usp18 mRNA; (D) Gbp-6 mRNA; (E) Ifnγ mRNA; (F) Gbp-1 mRNA (upper panel) and protein (lower panel); (G) Stat-1 mRNA (upper panel) and protein (lower panel); (H), Casp6 mRNA; (I) Casp12 mRNA; and (J) Afp mRNA. Expression of mRNA in control, shSep15, shTR1, and shTR1/Sep15 cells was determined by real-time RT-PCR, and expressed relative to Gapdh. Columns, mean (n = 3–6); bars, SE; (*P<0.05, **P<0.01, ***P<0.001). Protein expression was determined by Western blotting, and expressed relative to β-actin or α-tubulin, as indicated.

Mentions: The most strongly up-regulated gene signals in shSep15 cells were interferon-induced-protein-44 (Ifi44), ubiquitin-specific-peptidase-18 (Usp18), immunity-related-GTPase family-M-member-2 (Irgm2), interferon-regulatory-factor-7 (Irf7), interferon-γ-induced-GTPase (Igtp), very large interferon-inducible-GTPase (Gvin1), and guanylate-binding-proteins Gbp-1, -2 and -6. Significantly increased mRNA expression of Ifi44 (P<0.001, Fig 4A, upper panel), Irf7 (P<0.001, Fig 4B), and Usp18 (P<0.05, Fig 4C) in shSep15 cells compared to controls were validated with qPCR. Protein expression levels of Ifi44 (Fig 4A, lower panel), were slightly increased in shSep15 cells. The mRNA levels of Gbp-6 (P<0.05; Fig 4D) and interferon-γ (Ifnγ) (P<0.05; Fig 4E) were significantly higher in shSep15 compared to shTR1/shSep15 cells. The levels of Gbp-1 mRNA were previously reported to be highly up-regulated in Sep15 knockout mice [7]. Consistent with these observations, we found that Gbp-1 mRNA (Fig 4F, upper panel) and protein levels (Fig 4F, lower panel) were significantly increased exclusively in shSep15 cells. Ingenuity Pathway Analyses (IPA) indicated that inflammation-related genes up-regulated in shSep15 cells shared a common node in Stat-1 (S3A Fig), and included Gbp-2 (31.1-fold), Gbp-6 (30.1-fold), Nmi (2.8-fold), Irgm2 (20.7-fold), and Gbp-1 (14.3-fold; included in ‘Gbp-2*’). Other significantly up-regulated Stat-1-associated genes included Usp18 (65.1-fold), Irf7 (9.9-fold) and Irf9 (3.6-fold). Stat-1 mRNA was quantified with qPCR and found to be significantly increased only in shSep15 cells compared to controls (P<0.01, Fig 4G, upper panel), but protein levels of unphosphorylated Stat-1 remained unchanged (Fig 4G, lower panel). Stat-1 is thought to promote activation of different caspases, and the mRNA levels of caspase 6 (Fig 4H) and caspase 12 (Fig 4I) were significantly increased only in shSep15 cells (P<0.05). Supporting this pattern of involvement of interferon-γ and Sep15 are additional comparisons with our previously published microarray data [7], which showed a modest but significantly (P<0.05) increased mRNA expression of interferon-γ-regulated Stat-1, Stat-5A, Irf-2, and Irf-3, in Sep15 knockout mice (N = 4) compared to litter mate controls (N = 4). The gene most significantly down-regulated in shSep15 cells was alpha-fetoprotein (Afp). Interestingly, mRNA levels of Afp were increased 1.5-fold in shTR1/shSep15 cells as determined by qPCR (Fig 4J), but protein levels remained unchanged (S2A Fig).


The 15kDa selenoprotein and thioredoxin reductase 1 promote colon cancer by different pathways.

Tsuji PA, Carlson BA, Yoo MH, Naranjo-Suarez S, Xu XM, He Y, Asaki E, Seifried HE, Reinhold WC, Davis CD, Gladyshev VN, Hatfield DL - PLoS ONE (2015)

Validation of gene expression for genes significantly changed in shSep15 cells.Expression of (A) Ifi44 mRNA (upper panel) and protein (lower panel); (B) Irf-7 mRNA; (C) Usp18 mRNA; (D) Gbp-6 mRNA; (E) Ifnγ mRNA; (F) Gbp-1 mRNA (upper panel) and protein (lower panel); (G) Stat-1 mRNA (upper panel) and protein (lower panel); (H), Casp6 mRNA; (I) Casp12 mRNA; and (J) Afp mRNA. Expression of mRNA in control, shSep15, shTR1, and shTR1/Sep15 cells was determined by real-time RT-PCR, and expressed relative to Gapdh. Columns, mean (n = 3–6); bars, SE; (*P<0.05, **P<0.01, ***P<0.001). Protein expression was determined by Western blotting, and expressed relative to β-actin or α-tubulin, as indicated.
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Related In: Results  -  Collection

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pone.0124487.g004: Validation of gene expression for genes significantly changed in shSep15 cells.Expression of (A) Ifi44 mRNA (upper panel) and protein (lower panel); (B) Irf-7 mRNA; (C) Usp18 mRNA; (D) Gbp-6 mRNA; (E) Ifnγ mRNA; (F) Gbp-1 mRNA (upper panel) and protein (lower panel); (G) Stat-1 mRNA (upper panel) and protein (lower panel); (H), Casp6 mRNA; (I) Casp12 mRNA; and (J) Afp mRNA. Expression of mRNA in control, shSep15, shTR1, and shTR1/Sep15 cells was determined by real-time RT-PCR, and expressed relative to Gapdh. Columns, mean (n = 3–6); bars, SE; (*P<0.05, **P<0.01, ***P<0.001). Protein expression was determined by Western blotting, and expressed relative to β-actin or α-tubulin, as indicated.
Mentions: The most strongly up-regulated gene signals in shSep15 cells were interferon-induced-protein-44 (Ifi44), ubiquitin-specific-peptidase-18 (Usp18), immunity-related-GTPase family-M-member-2 (Irgm2), interferon-regulatory-factor-7 (Irf7), interferon-γ-induced-GTPase (Igtp), very large interferon-inducible-GTPase (Gvin1), and guanylate-binding-proteins Gbp-1, -2 and -6. Significantly increased mRNA expression of Ifi44 (P<0.001, Fig 4A, upper panel), Irf7 (P<0.001, Fig 4B), and Usp18 (P<0.05, Fig 4C) in shSep15 cells compared to controls were validated with qPCR. Protein expression levels of Ifi44 (Fig 4A, lower panel), were slightly increased in shSep15 cells. The mRNA levels of Gbp-6 (P<0.05; Fig 4D) and interferon-γ (Ifnγ) (P<0.05; Fig 4E) were significantly higher in shSep15 compared to shTR1/shSep15 cells. The levels of Gbp-1 mRNA were previously reported to be highly up-regulated in Sep15 knockout mice [7]. Consistent with these observations, we found that Gbp-1 mRNA (Fig 4F, upper panel) and protein levels (Fig 4F, lower panel) were significantly increased exclusively in shSep15 cells. Ingenuity Pathway Analyses (IPA) indicated that inflammation-related genes up-regulated in shSep15 cells shared a common node in Stat-1 (S3A Fig), and included Gbp-2 (31.1-fold), Gbp-6 (30.1-fold), Nmi (2.8-fold), Irgm2 (20.7-fold), and Gbp-1 (14.3-fold; included in ‘Gbp-2*’). Other significantly up-regulated Stat-1-associated genes included Usp18 (65.1-fold), Irf7 (9.9-fold) and Irf9 (3.6-fold). Stat-1 mRNA was quantified with qPCR and found to be significantly increased only in shSep15 cells compared to controls (P<0.01, Fig 4G, upper panel), but protein levels of unphosphorylated Stat-1 remained unchanged (Fig 4G, lower panel). Stat-1 is thought to promote activation of different caspases, and the mRNA levels of caspase 6 (Fig 4H) and caspase 12 (Fig 4I) were significantly increased only in shSep15 cells (P<0.05). Supporting this pattern of involvement of interferon-γ and Sep15 are additional comparisons with our previously published microarray data [7], which showed a modest but significantly (P<0.05) increased mRNA expression of interferon-γ-regulated Stat-1, Stat-5A, Irf-2, and Irf-3, in Sep15 knockout mice (N = 4) compared to litter mate controls (N = 4). The gene most significantly down-regulated in shSep15 cells was alpha-fetoprotein (Afp). Interestingly, mRNA levels of Afp were increased 1.5-fold in shTR1/shSep15 cells as determined by qPCR (Fig 4J), but protein levels remained unchanged (S2A Fig).

Bottom Line: We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells.These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells.Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Towson University, Towson, Maryland, United States of America.

ABSTRACT
Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

No MeSH data available.


Related in: MedlinePlus