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Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers.

He S, Zhao Z, Yang Y, O'Connell D, Zhang X, Oh S, Ma B, Lee JH, Zhang T, Varghese B, Yip J, Dolatshahi Pirooz S, Li M, Zhang Y, Li GM, Ellen Martin S, Machida K, Liang C - Nat Commun (2015)

Bottom Line: However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive.Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect.These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck Medical School, University of Southern California, Los Angeles, California 90033, USA.

ABSTRACT
Autophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

No MeSH data available.


Related in: MedlinePlus

UVRAGFS promotes chromosomal instability and centrosome amplification.(a) Representative SKY analysis of mouse embryonic stem (ES) cells expressing vector or Flag-UVRAGFS. Average chromosomal aberrations per cell were quantified. *P<0.05, Wilcoxon Signed-rank Test. (b) Chromosomal rearrangement and single-nucleotide variants (SNVs) in UVRAGFS gastric cancer. Representative Circos plots of the UVRAGWT and UVRAGFS subtypes of MSI gastric cancer in the Pfizer and UHK cohorts20. The inner circle denotes chromosomal structural variants: ITX (intrachromosomal translocation), blue; DEL (deletion), red; CTX (interchromosomal translocation), green; INV (inversions), pink; Tandem duplication, black. In the second circle, each dot denotes one somatic SNV, coloured according to six mutation types: T>G, pink; T>C, green; T>A, gray; C>G, black; C>A, blue; C>T, red. The outer circle denotes 23 chromosomes. Boxplot represents the total chromosomal structure variants (SV) count in the UVRAGWT and UVRAGFS MSI gastric cancers (Mann–Whitney test). (c) UVRAGFS induces centrosome amplification. SW480.Vector and SW480.UVRAGFS cells with different centrosome numbers were immunostained for γ-Tubulin and DAPI and quantified (Data are the means±s.d., n=200 cells obtained by gathering data from three independent experiments). (c) centrosome. Scale bar, 10 μm. (d) Representative confocal images of spindle malformation in mitotic Vector, UVRAGWT, and UVRAGFS SW480 cells co-stained with anti-γ-Tubulin (red) and anti-α-Tubulin (green) for the mitotic asters. The percentages of cells with disorganized spindle were quantified. Data are the means±s.d. (n=200 cells obtained by pooling data from three independent experiments). Scale bar, 10 μm. (e) UVRAGFS is defective in CEP63 binding. Whole-cell lysates (WCL) of 293T transfected with Flag-UVRAGWT or Flag-UVRAGFS were immunoprecipitated with anti-Flag followed by IB with anti-CEP63. (f) Representative image showing dissociation of UVRAGFS from the centrosome. HeLa cells expressing Flag-UVRAGWT or Flag-UVRAGFS were stained with anti-Flag (green), anti-γ-Tubulin (red), and anti-CEP63 (blue). Scale bar, 10 μm. (g) UVRAGFS inhibits UVRAG-CEP63 interaction. The 293T cells were transfected with increasing amounts of Flag-UVRAGFS. WCL were immunoprecipitated with anti-CEP63 or anti-Flag, followed by immunoblotting with anti-UVRAG or anti-CEP63 as indicated.
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f4: UVRAGFS promotes chromosomal instability and centrosome amplification.(a) Representative SKY analysis of mouse embryonic stem (ES) cells expressing vector or Flag-UVRAGFS. Average chromosomal aberrations per cell were quantified. *P<0.05, Wilcoxon Signed-rank Test. (b) Chromosomal rearrangement and single-nucleotide variants (SNVs) in UVRAGFS gastric cancer. Representative Circos plots of the UVRAGWT and UVRAGFS subtypes of MSI gastric cancer in the Pfizer and UHK cohorts20. The inner circle denotes chromosomal structural variants: ITX (intrachromosomal translocation), blue; DEL (deletion), red; CTX (interchromosomal translocation), green; INV (inversions), pink; Tandem duplication, black. In the second circle, each dot denotes one somatic SNV, coloured according to six mutation types: T>G, pink; T>C, green; T>A, gray; C>G, black; C>A, blue; C>T, red. The outer circle denotes 23 chromosomes. Boxplot represents the total chromosomal structure variants (SV) count in the UVRAGWT and UVRAGFS MSI gastric cancers (Mann–Whitney test). (c) UVRAGFS induces centrosome amplification. SW480.Vector and SW480.UVRAGFS cells with different centrosome numbers were immunostained for γ-Tubulin and DAPI and quantified (Data are the means±s.d., n=200 cells obtained by gathering data from three independent experiments). (c) centrosome. Scale bar, 10 μm. (d) Representative confocal images of spindle malformation in mitotic Vector, UVRAGWT, and UVRAGFS SW480 cells co-stained with anti-γ-Tubulin (red) and anti-α-Tubulin (green) for the mitotic asters. The percentages of cells with disorganized spindle were quantified. Data are the means±s.d. (n=200 cells obtained by pooling data from three independent experiments). Scale bar, 10 μm. (e) UVRAGFS is defective in CEP63 binding. Whole-cell lysates (WCL) of 293T transfected with Flag-UVRAGWT or Flag-UVRAGFS were immunoprecipitated with anti-Flag followed by IB with anti-CEP63. (f) Representative image showing dissociation of UVRAGFS from the centrosome. HeLa cells expressing Flag-UVRAGWT or Flag-UVRAGFS were stained with anti-Flag (green), anti-γ-Tubulin (red), and anti-CEP63 (blue). Scale bar, 10 μm. (g) UVRAGFS inhibits UVRAG-CEP63 interaction. The 293T cells were transfected with increasing amounts of Flag-UVRAGFS. WCL were immunoprecipitated with anti-CEP63 or anti-Flag, followed by immunoblotting with anti-UVRAG or anti-CEP63 as indicated.

Mentions: Because the role of UVRAG in cancer has been linked to its ability to maintain chromosomal stability17, we investigated the effect of UVRAGFS on overall chromosomal stability in genetically stable mouse embryonic stem cells. Spectral karyotyping analysis showed that, unlike control cells that were mostly diploid, UVRAGFS-embryonic stem cells were highly heterogeneous with respect to both structural and numerical aberrations as compared with the vector control (Fig. 4a, Supplementary Fig. 4a) with a greater than sevenfold increase in aneuploidy in UVRAGFS cells (Supplementary Fig. 4b). These results indicate that UVRAGFS elicits severe chromosomal instability and aneuploidy. To validate this, we analysed the Pfizer and UHK cohort20 of gastric cancers, and observed significantly enhanced chromosomal rearrangement in UVRAGFS MSI gastric cancers as compared with UVRAGWT MSI gastric cancers (Fig. 4b). In fact, UVRAGFS gastric cancers had substantially more protein-altering mutations and single-nucleotide variants than UVRAGWT MSI and MSS gastric cancers (Supplementary Fig. 4c). Moreover, the FS mutation appeared to be more frequent in gastric cases with advanced tumour, node, metastasis stage (Supplementary Fig. 4d). Thus, UVRAGFS may predispose MSI cancers to increased genetic instability and cancer progression.


Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers.

He S, Zhao Z, Yang Y, O'Connell D, Zhang X, Oh S, Ma B, Lee JH, Zhang T, Varghese B, Yip J, Dolatshahi Pirooz S, Li M, Zhang Y, Li GM, Ellen Martin S, Machida K, Liang C - Nat Commun (2015)

UVRAGFS promotes chromosomal instability and centrosome amplification.(a) Representative SKY analysis of mouse embryonic stem (ES) cells expressing vector or Flag-UVRAGFS. Average chromosomal aberrations per cell were quantified. *P<0.05, Wilcoxon Signed-rank Test. (b) Chromosomal rearrangement and single-nucleotide variants (SNVs) in UVRAGFS gastric cancer. Representative Circos plots of the UVRAGWT and UVRAGFS subtypes of MSI gastric cancer in the Pfizer and UHK cohorts20. The inner circle denotes chromosomal structural variants: ITX (intrachromosomal translocation), blue; DEL (deletion), red; CTX (interchromosomal translocation), green; INV (inversions), pink; Tandem duplication, black. In the second circle, each dot denotes one somatic SNV, coloured according to six mutation types: T>G, pink; T>C, green; T>A, gray; C>G, black; C>A, blue; C>T, red. The outer circle denotes 23 chromosomes. Boxplot represents the total chromosomal structure variants (SV) count in the UVRAGWT and UVRAGFS MSI gastric cancers (Mann–Whitney test). (c) UVRAGFS induces centrosome amplification. SW480.Vector and SW480.UVRAGFS cells with different centrosome numbers were immunostained for γ-Tubulin and DAPI and quantified (Data are the means±s.d., n=200 cells obtained by gathering data from three independent experiments). (c) centrosome. Scale bar, 10 μm. (d) Representative confocal images of spindle malformation in mitotic Vector, UVRAGWT, and UVRAGFS SW480 cells co-stained with anti-γ-Tubulin (red) and anti-α-Tubulin (green) for the mitotic asters. The percentages of cells with disorganized spindle were quantified. Data are the means±s.d. (n=200 cells obtained by pooling data from three independent experiments). Scale bar, 10 μm. (e) UVRAGFS is defective in CEP63 binding. Whole-cell lysates (WCL) of 293T transfected with Flag-UVRAGWT or Flag-UVRAGFS were immunoprecipitated with anti-Flag followed by IB with anti-CEP63. (f) Representative image showing dissociation of UVRAGFS from the centrosome. HeLa cells expressing Flag-UVRAGWT or Flag-UVRAGFS were stained with anti-Flag (green), anti-γ-Tubulin (red), and anti-CEP63 (blue). Scale bar, 10 μm. (g) UVRAGFS inhibits UVRAG-CEP63 interaction. The 293T cells were transfected with increasing amounts of Flag-UVRAGFS. WCL were immunoprecipitated with anti-CEP63 or anti-Flag, followed by immunoblotting with anti-UVRAG or anti-CEP63 as indicated.
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f4: UVRAGFS promotes chromosomal instability and centrosome amplification.(a) Representative SKY analysis of mouse embryonic stem (ES) cells expressing vector or Flag-UVRAGFS. Average chromosomal aberrations per cell were quantified. *P<0.05, Wilcoxon Signed-rank Test. (b) Chromosomal rearrangement and single-nucleotide variants (SNVs) in UVRAGFS gastric cancer. Representative Circos plots of the UVRAGWT and UVRAGFS subtypes of MSI gastric cancer in the Pfizer and UHK cohorts20. The inner circle denotes chromosomal structural variants: ITX (intrachromosomal translocation), blue; DEL (deletion), red; CTX (interchromosomal translocation), green; INV (inversions), pink; Tandem duplication, black. In the second circle, each dot denotes one somatic SNV, coloured according to six mutation types: T>G, pink; T>C, green; T>A, gray; C>G, black; C>A, blue; C>T, red. The outer circle denotes 23 chromosomes. Boxplot represents the total chromosomal structure variants (SV) count in the UVRAGWT and UVRAGFS MSI gastric cancers (Mann–Whitney test). (c) UVRAGFS induces centrosome amplification. SW480.Vector and SW480.UVRAGFS cells with different centrosome numbers were immunostained for γ-Tubulin and DAPI and quantified (Data are the means±s.d., n=200 cells obtained by gathering data from three independent experiments). (c) centrosome. Scale bar, 10 μm. (d) Representative confocal images of spindle malformation in mitotic Vector, UVRAGWT, and UVRAGFS SW480 cells co-stained with anti-γ-Tubulin (red) and anti-α-Tubulin (green) for the mitotic asters. The percentages of cells with disorganized spindle were quantified. Data are the means±s.d. (n=200 cells obtained by pooling data from three independent experiments). Scale bar, 10 μm. (e) UVRAGFS is defective in CEP63 binding. Whole-cell lysates (WCL) of 293T transfected with Flag-UVRAGWT or Flag-UVRAGFS were immunoprecipitated with anti-Flag followed by IB with anti-CEP63. (f) Representative image showing dissociation of UVRAGFS from the centrosome. HeLa cells expressing Flag-UVRAGWT or Flag-UVRAGFS were stained with anti-Flag (green), anti-γ-Tubulin (red), and anti-CEP63 (blue). Scale bar, 10 μm. (g) UVRAGFS inhibits UVRAG-CEP63 interaction. The 293T cells were transfected with increasing amounts of Flag-UVRAGFS. WCL were immunoprecipitated with anti-CEP63 or anti-Flag, followed by immunoblotting with anti-UVRAG or anti-CEP63 as indicated.
Mentions: Because the role of UVRAG in cancer has been linked to its ability to maintain chromosomal stability17, we investigated the effect of UVRAGFS on overall chromosomal stability in genetically stable mouse embryonic stem cells. Spectral karyotyping analysis showed that, unlike control cells that were mostly diploid, UVRAGFS-embryonic stem cells were highly heterogeneous with respect to both structural and numerical aberrations as compared with the vector control (Fig. 4a, Supplementary Fig. 4a) with a greater than sevenfold increase in aneuploidy in UVRAGFS cells (Supplementary Fig. 4b). These results indicate that UVRAGFS elicits severe chromosomal instability and aneuploidy. To validate this, we analysed the Pfizer and UHK cohort20 of gastric cancers, and observed significantly enhanced chromosomal rearrangement in UVRAGFS MSI gastric cancers as compared with UVRAGWT MSI gastric cancers (Fig. 4b). In fact, UVRAGFS gastric cancers had substantially more protein-altering mutations and single-nucleotide variants than UVRAGWT MSI and MSS gastric cancers (Supplementary Fig. 4c). Moreover, the FS mutation appeared to be more frequent in gastric cases with advanced tumour, node, metastasis stage (Supplementary Fig. 4d). Thus, UVRAGFS may predispose MSI cancers to increased genetic instability and cancer progression.

Bottom Line: However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive.Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect.These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck Medical School, University of Southern California, Los Angeles, California 90033, USA.

ABSTRACT
Autophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

No MeSH data available.


Related in: MedlinePlus