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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 sensitizes CRC to DNA damage-inducing chemotherapy.(a) Mice bearing SW480.Vec and SW480.UVRAGFS tumours were treated with saline or 5-FU over day 1–30 when the tumour volume reached 200 mm3. Mean relative tumour volume (n=10), is expressed compared with tumour volumes on day 1. *P<0.05; **P<0.01; ***P<0.001. (b) Relative tumour volume for individual mice treated in a at day 25. (c) Representative images of mice bearing SW480-xenografts on the day 0, 21–28 of chemotherapy (left). Western blots showed UVRAGFS expression in representative tumours. Scale bar, 5 mm. (d) Immunohistochemical analysis of SW480-xenografts harvested from mice treated for 28 continuous days with 5-FU. Representative sections were stained (left) as indicated and staining-positive cells were quantified (right) as means±s.d. Scale bar, 10 μm. *P<0.05; **P<0.01; ***P<0.001. (e) SW480.Vector, SW480.UVRAGWT and SW480.UVRAGFS were treated with the indicated doses of 5-FU, Oxaliplatin and Irinotecan, followed by colony survival assay. Data are the means±s.d. (n=3). *P<0.05; **P<0.01.
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f6: UVRAGFS sensitizes CRC to DNA damage-inducing chemotherapy.(a) Mice bearing SW480.Vec and SW480.UVRAGFS tumours were treated with saline or 5-FU over day 1–30 when the tumour volume reached 200 mm3. Mean relative tumour volume (n=10), is expressed compared with tumour volumes on day 1. *P<0.05; **P<0.01; ***P<0.001. (b) Relative tumour volume for individual mice treated in a at day 25. (c) Representative images of mice bearing SW480-xenografts on the day 0, 21–28 of chemotherapy (left). Western blots showed UVRAGFS expression in representative tumours. Scale bar, 5 mm. (d) Immunohistochemical analysis of SW480-xenografts harvested from mice treated for 28 continuous days with 5-FU. Representative sections were stained (left) as indicated and staining-positive cells were quantified (right) as means±s.d. Scale bar, 10 μm. *P<0.05; **P<0.01; ***P<0.001. (e) SW480.Vector, SW480.UVRAGWT and SW480.UVRAGFS were treated with the indicated doses of 5-FU, Oxaliplatin and Irinotecan, followed by colony survival assay. Data are the means±s.d. (n=3). *P<0.05; **P<0.01.

Mentions: We next investigated the possible clinical relevance of UVRAGFS by testing the response of CRC to 5-FU chemotherapy, the first-line treatment for CRC patients, using a tumour xenograft model. Surprisingly, UVRAGFS expression significantly increased tumour sensitivity to 5-FU treatment with an approximate 10-fold reduction in tumour volumes after a 4-week administration of 5-FU (Fig. 6a), compared with a less than twofold reduction in the control group (Fig. 6a–c). Histological analyses revealed a significant reduction in cell proliferation and an increase in the number of cells undergoing apoptosis in 5-FU-treated UVRAGFS tumours, in concordance with induced tumour shrinkage (Fig. 6d). In addition, UVRAGFS expression in CRC cells markedly increased their sensitivity to other DNA-based cytotoxic anticancer agents, including oxaliplatin and irinotecan, as shown by reduced rates of clonogenic survival, whereas UVRAGWT cells were resistant to the drugs (Fig. 6e). To examine the unexpected role of UVRAGFS in tumour chemosensitivity, we measured the levels of γ-H2AX, a sensitive marker of double strand breaks (DSBs)35, and observed that UVRAGFS SW480-tumours accumulated higher levels of γ-H2AX than the controls, which further increased with 5-FU that produces DNA strand breaks (Fig. 6d). Consistent with our observation in xenograft tumours, UVRAGFS expression resulted in a significant increase of γ-H2AX foci and levels in SW480 CRC cells (Supplementary Fig. 7a,b). Furthermore, the overall levels of γ-H2AX were higher in MSI CRC cell lines expressing UVRAGFS compared with the WT counterparts, and likewise, were significantly different between UVRAGFS-positive and -negative primary tumours (Fig. 1b,d). Adding UVRAGWT to UVRAGFS-positive HCT116 and RKO cells at different doses clearly suppressed the levels of DSBs (Supplementary Fig. 7c), highlighting a direct involvement of UVRAGFS in genetic stability. To determine whether the observed accumulation of DSB in UVRAGFS cells reflects impaired DNA repair, we measured unrepaired DSBs after ionizing radiation (IR) using the comet assay. We found that IR induced comparable levels of DNA damage in vector, UVRAGWT and UVRAGFS cells (10 min post-IR in Fig. 7a). However, a high persistence of comet tails was observed 24 h post-irradiation in UVRAGFS cells, whereas UVRAGWT cells have repaired most of the damaged DNA. These data indicate that UVRAGFS disrupts the rapid repair process of DSBs. The inhibitory effect of UVRAGFS on DSB repair was also detected in the autophagy-competent Atg3+/+ and the autophagy- Atg3−/− cells (Supplementary Fig. 7d), suggesting minimal participation of autophagy in the elevated DNA damage induced by UVRAGFS expression.


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 sensitizes CRC to DNA damage-inducing chemotherapy.(a) Mice bearing SW480.Vec and SW480.UVRAGFS tumours were treated with saline or 5-FU over day 1–30 when the tumour volume reached 200 mm3. Mean relative tumour volume (n=10), is expressed compared with tumour volumes on day 1. *P<0.05; **P<0.01; ***P<0.001. (b) Relative tumour volume for individual mice treated in a at day 25. (c) Representative images of mice bearing SW480-xenografts on the day 0, 21–28 of chemotherapy (left). Western blots showed UVRAGFS expression in representative tumours. Scale bar, 5 mm. (d) Immunohistochemical analysis of SW480-xenografts harvested from mice treated for 28 continuous days with 5-FU. Representative sections were stained (left) as indicated and staining-positive cells were quantified (right) as means±s.d. Scale bar, 10 μm. *P<0.05; **P<0.01; ***P<0.001. (e) SW480.Vector, SW480.UVRAGWT and SW480.UVRAGFS were treated with the indicated doses of 5-FU, Oxaliplatin and Irinotecan, followed by colony survival assay. Data are the means±s.d. (n=3). *P<0.05; **P<0.01.
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f6: UVRAGFS sensitizes CRC to DNA damage-inducing chemotherapy.(a) Mice bearing SW480.Vec and SW480.UVRAGFS tumours were treated with saline or 5-FU over day 1–30 when the tumour volume reached 200 mm3. Mean relative tumour volume (n=10), is expressed compared with tumour volumes on day 1. *P<0.05; **P<0.01; ***P<0.001. (b) Relative tumour volume for individual mice treated in a at day 25. (c) Representative images of mice bearing SW480-xenografts on the day 0, 21–28 of chemotherapy (left). Western blots showed UVRAGFS expression in representative tumours. Scale bar, 5 mm. (d) Immunohistochemical analysis of SW480-xenografts harvested from mice treated for 28 continuous days with 5-FU. Representative sections were stained (left) as indicated and staining-positive cells were quantified (right) as means±s.d. Scale bar, 10 μm. *P<0.05; **P<0.01; ***P<0.001. (e) SW480.Vector, SW480.UVRAGWT and SW480.UVRAGFS were treated with the indicated doses of 5-FU, Oxaliplatin and Irinotecan, followed by colony survival assay. Data are the means±s.d. (n=3). *P<0.05; **P<0.01.
Mentions: We next investigated the possible clinical relevance of UVRAGFS by testing the response of CRC to 5-FU chemotherapy, the first-line treatment for CRC patients, using a tumour xenograft model. Surprisingly, UVRAGFS expression significantly increased tumour sensitivity to 5-FU treatment with an approximate 10-fold reduction in tumour volumes after a 4-week administration of 5-FU (Fig. 6a), compared with a less than twofold reduction in the control group (Fig. 6a–c). Histological analyses revealed a significant reduction in cell proliferation and an increase in the number of cells undergoing apoptosis in 5-FU-treated UVRAGFS tumours, in concordance with induced tumour shrinkage (Fig. 6d). In addition, UVRAGFS expression in CRC cells markedly increased their sensitivity to other DNA-based cytotoxic anticancer agents, including oxaliplatin and irinotecan, as shown by reduced rates of clonogenic survival, whereas UVRAGWT cells were resistant to the drugs (Fig. 6e). To examine the unexpected role of UVRAGFS in tumour chemosensitivity, we measured the levels of γ-H2AX, a sensitive marker of double strand breaks (DSBs)35, and observed that UVRAGFS SW480-tumours accumulated higher levels of γ-H2AX than the controls, which further increased with 5-FU that produces DNA strand breaks (Fig. 6d). Consistent with our observation in xenograft tumours, UVRAGFS expression resulted in a significant increase of γ-H2AX foci and levels in SW480 CRC cells (Supplementary Fig. 7a,b). Furthermore, the overall levels of γ-H2AX were higher in MSI CRC cell lines expressing UVRAGFS compared with the WT counterparts, and likewise, were significantly different between UVRAGFS-positive and -negative primary tumours (Fig. 1b,d). Adding UVRAGWT to UVRAGFS-positive HCT116 and RKO cells at different doses clearly suppressed the levels of DSBs (Supplementary Fig. 7c), highlighting a direct involvement of UVRAGFS in genetic stability. To determine whether the observed accumulation of DSB in UVRAGFS cells reflects impaired DNA repair, we measured unrepaired DSBs after ionizing radiation (IR) using the comet assay. We found that IR induced comparable levels of DNA damage in vector, UVRAGWT and UVRAGFS cells (10 min post-IR in Fig. 7a). However, a high persistence of comet tails was observed 24 h post-irradiation in UVRAGFS cells, whereas UVRAGWT cells have repaired most of the damaged DNA. These data indicate that UVRAGFS disrupts the rapid repair process of DSBs. The inhibitory effect of UVRAGFS on DSB repair was also detected in the autophagy-competent Atg3+/+ and the autophagy- Atg3−/− cells (Supplementary Fig. 7d), suggesting minimal participation of autophagy in the elevated DNA damage induced by UVRAGFS expression.

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