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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

Identification of UVRAG FS mutation in CRC cell lines and primary tumours.(a) Sequencing analysis of UVRAG at the location of the A10 repeat in MSS (HCC2998, COLO205, SW620, SW480 and HT29) and MSI (HCT15, SW48, HCT116, RKO, LIM2405, LS180 and KM12) CRC cell lines. Arrows indicate the heterozygous deletion of one A in UVRAG A10 in MSI cell lines. (b,c) Wild-type (WT) and FS mutant UVRAG protein expression in MSS and MSI CRC cell lines. Whole-cell lysates (WCL) of MSS and MSI CRC cell lines were immunoprecipitated with anti-UVRAGFS followed by immunoblotting with anti-UVRAGFS, or they were directly probed with antibodies targeting UVRAGWT or γ-H2AX. Actin served as a loading control. Densitometric quantification of protein expression is shown in (c). Dash lines indicate average band intensities of all the tested cell lines. Note reduced UVRAGWT expression in MSI CRC cells expressing UVRAGFS. (d) H&E (first row) and immunohistochemical analysis of UVRAG (second row), Ki67 (fourth row), and γ-H2AX (5th row) in paired human primary CRC specimen obtained from three separate patients with their corresponding status of UVRAG FS mutation (third row) provided. The bar plots (right) are the quantification of the levels of Ki67 and γ-H2AX (denoted by arrows) in the paired tissues with WT or mutant UVRAG. HPF, high-power field. ***P<0.001 (Mann–Whitney test); Scale bar ,50 μm.
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f1: Identification of UVRAG FS mutation in CRC cell lines and primary tumours.(a) Sequencing analysis of UVRAG at the location of the A10 repeat in MSS (HCC2998, COLO205, SW620, SW480 and HT29) and MSI (HCT15, SW48, HCT116, RKO, LIM2405, LS180 and KM12) CRC cell lines. Arrows indicate the heterozygous deletion of one A in UVRAG A10 in MSI cell lines. (b,c) Wild-type (WT) and FS mutant UVRAG protein expression in MSS and MSI CRC cell lines. Whole-cell lysates (WCL) of MSS and MSI CRC cell lines were immunoprecipitated with anti-UVRAGFS followed by immunoblotting with anti-UVRAGFS, or they were directly probed with antibodies targeting UVRAGWT or γ-H2AX. Actin served as a loading control. Densitometric quantification of protein expression is shown in (c). Dash lines indicate average band intensities of all the tested cell lines. Note reduced UVRAGWT expression in MSI CRC cells expressing UVRAGFS. (d) H&E (first row) and immunohistochemical analysis of UVRAG (second row), Ki67 (fourth row), and γ-H2AX (5th row) in paired human primary CRC specimen obtained from three separate patients with their corresponding status of UVRAG FS mutation (third row) provided. The bar plots (right) are the quantification of the levels of Ki67 and γ-H2AX (denoted by arrows) in the paired tissues with WT or mutant UVRAG. HPF, high-power field. ***P<0.001 (Mann–Whitney test); Scale bar ,50 μm.

Mentions: The human UVRAG gene contains a tract of A10 mononucleotide repeats in exon 8, spanning codons 234–237 (5′-AAA AAA AAA AGT-3′; Supplementary Fig. 1a,b). Using seven MSI+ CRC cell lines (HCT15, HCT116, KM12, LIM2405, LS180, RKO and SW48) and genomic sequencing, we confirmed, as reported previously6716, the heterozygous FS deletion of one nucleotide (A) in the UVRAG A10-coding repeat in most tested MSI+ CRC cells, with the exception of HCT15 and SW48. In contrast, MSS (microsatellite stable) cells, including COLO205, HCC2998, HT29, SW480 and SW620, contained only WT coding repeats (Fig. 1a). The FS mutation was predicted to produce a premature stop codon and therefore a truncated UVRAG7 (referred here as UVRAGFS; Supplementary Fig. 1a,b). To assess whether this mutation is indeed expressed in MSI cells, we generated an antibody specifically recognizing UVRAGFS, but not UVRAGWT, using the FS-derived neopeptide (234KKKVNACS241) as antigen (Supplementary Fig. 1b,c). UVRAGFS expression was detected in all MSI cell lines carrying the FS mutation, but not in MSI or MSS cells that are WT for UVRAG (Fig. 1b). Notably, the overall expression of UVRAGWT was diminished in MSI cells with the FS mutation (Fig. 1b), and the levels of UVRAGFS were inversely correlated with the expression of UVRAGWT in all tested cell lines (Fig. 1c). This was consistent with the UVRAG expression profile from the CRC cell lines of the NCI-60 panel19. Therein, a significant reduction of UVRAGWT expression was detected in UVRAGFS-positive KM12 and HCT116 CRC cells compared with other CRC cells without UVRAGFS (Supplementary Fig. 1d). In addition, the UVRAG FS mutation was present in one of the four analysed cases of human primary CRC with MSI (fourth column in Fig. 1d), but not in primary MSS CRC or in normal colorectal mucosa (Fig. 1d, Supplementary Table 1). This is in line with a previous report2α that evaluated the mutation frequencies in 137 genes in MSI cancers, revealing the high frequency of the A10UVRAG FS mutation that was found in 33% CRC, 8% endometrial and 7.8% gastric cancers with MSI (Supplementary Fig. 1e). Whole-genome sequencing analysis of a large cohort of gastric cancers (Pfizer and UHK; n=100) also confirmed the presence of the UVRAG FS mutation in MSI gastric cancer (40%)20. Collectively, these results indicate that the frameshift UVRAG mutation is likely selected and is expressed as a truncated UVRAG protein in MSI tumours.


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)

Identification of UVRAG FS mutation in CRC cell lines and primary tumours.(a) Sequencing analysis of UVRAG at the location of the A10 repeat in MSS (HCC2998, COLO205, SW620, SW480 and HT29) and MSI (HCT15, SW48, HCT116, RKO, LIM2405, LS180 and KM12) CRC cell lines. Arrows indicate the heterozygous deletion of one A in UVRAG A10 in MSI cell lines. (b,c) Wild-type (WT) and FS mutant UVRAG protein expression in MSS and MSI CRC cell lines. Whole-cell lysates (WCL) of MSS and MSI CRC cell lines were immunoprecipitated with anti-UVRAGFS followed by immunoblotting with anti-UVRAGFS, or they were directly probed with antibodies targeting UVRAGWT or γ-H2AX. Actin served as a loading control. Densitometric quantification of protein expression is shown in (c). Dash lines indicate average band intensities of all the tested cell lines. Note reduced UVRAGWT expression in MSI CRC cells expressing UVRAGFS. (d) H&E (first row) and immunohistochemical analysis of UVRAG (second row), Ki67 (fourth row), and γ-H2AX (5th row) in paired human primary CRC specimen obtained from three separate patients with their corresponding status of UVRAG FS mutation (third row) provided. The bar plots (right) are the quantification of the levels of Ki67 and γ-H2AX (denoted by arrows) in the paired tissues with WT or mutant UVRAG. HPF, high-power field. ***P<0.001 (Mann–Whitney test); Scale bar ,50 μm.
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f1: Identification of UVRAG FS mutation in CRC cell lines and primary tumours.(a) Sequencing analysis of UVRAG at the location of the A10 repeat in MSS (HCC2998, COLO205, SW620, SW480 and HT29) and MSI (HCT15, SW48, HCT116, RKO, LIM2405, LS180 and KM12) CRC cell lines. Arrows indicate the heterozygous deletion of one A in UVRAG A10 in MSI cell lines. (b,c) Wild-type (WT) and FS mutant UVRAG protein expression in MSS and MSI CRC cell lines. Whole-cell lysates (WCL) of MSS and MSI CRC cell lines were immunoprecipitated with anti-UVRAGFS followed by immunoblotting with anti-UVRAGFS, or they were directly probed with antibodies targeting UVRAGWT or γ-H2AX. Actin served as a loading control. Densitometric quantification of protein expression is shown in (c). Dash lines indicate average band intensities of all the tested cell lines. Note reduced UVRAGWT expression in MSI CRC cells expressing UVRAGFS. (d) H&E (first row) and immunohistochemical analysis of UVRAG (second row), Ki67 (fourth row), and γ-H2AX (5th row) in paired human primary CRC specimen obtained from three separate patients with their corresponding status of UVRAG FS mutation (third row) provided. The bar plots (right) are the quantification of the levels of Ki67 and γ-H2AX (denoted by arrows) in the paired tissues with WT or mutant UVRAG. HPF, high-power field. ***P<0.001 (Mann–Whitney test); Scale bar ,50 μm.
Mentions: The human UVRAG gene contains a tract of A10 mononucleotide repeats in exon 8, spanning codons 234–237 (5′-AAA AAA AAA AGT-3′; Supplementary Fig. 1a,b). Using seven MSI+ CRC cell lines (HCT15, HCT116, KM12, LIM2405, LS180, RKO and SW48) and genomic sequencing, we confirmed, as reported previously6716, the heterozygous FS deletion of one nucleotide (A) in the UVRAG A10-coding repeat in most tested MSI+ CRC cells, with the exception of HCT15 and SW48. In contrast, MSS (microsatellite stable) cells, including COLO205, HCC2998, HT29, SW480 and SW620, contained only WT coding repeats (Fig. 1a). The FS mutation was predicted to produce a premature stop codon and therefore a truncated UVRAG7 (referred here as UVRAGFS; Supplementary Fig. 1a,b). To assess whether this mutation is indeed expressed in MSI cells, we generated an antibody specifically recognizing UVRAGFS, but not UVRAGWT, using the FS-derived neopeptide (234KKKVNACS241) as antigen (Supplementary Fig. 1b,c). UVRAGFS expression was detected in all MSI cell lines carrying the FS mutation, but not in MSI or MSS cells that are WT for UVRAG (Fig. 1b). Notably, the overall expression of UVRAGWT was diminished in MSI cells with the FS mutation (Fig. 1b), and the levels of UVRAGFS were inversely correlated with the expression of UVRAGWT in all tested cell lines (Fig. 1c). This was consistent with the UVRAG expression profile from the CRC cell lines of the NCI-60 panel19. Therein, a significant reduction of UVRAGWT expression was detected in UVRAGFS-positive KM12 and HCT116 CRC cells compared with other CRC cells without UVRAGFS (Supplementary Fig. 1d). In addition, the UVRAG FS mutation was present in one of the four analysed cases of human primary CRC with MSI (fourth column in Fig. 1d), but not in primary MSS CRC or in normal colorectal mucosa (Fig. 1d, Supplementary Table 1). This is in line with a previous report2α that evaluated the mutation frequencies in 137 genes in MSI cancers, revealing the high frequency of the A10UVRAG FS mutation that was found in 33% CRC, 8% endometrial and 7.8% gastric cancers with MSI (Supplementary Fig. 1e). Whole-genome sequencing analysis of a large cohort of gastric cancers (Pfizer and UHK; n=100) also confirmed the presence of the UVRAG FS mutation in MSI gastric cancer (40%)20. Collectively, these results indicate that the frameshift UVRAG mutation is likely selected and is expressed as a truncated UVRAG protein in MSI tumours.

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