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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 inhibits cellular autophagy in a dominant-negative manner.(a) NIH3T3 cells stably expressing vector, UVRAGWT, and UVRAGFS were transfected with GFP–LC3 and treated with rapamycin (100 nM). GFP–LC3 puncta per cell were quantified as shown in representative images shown. Data represent the means±s.d. (n=6). *P<0.05. Scale bar, 10 μm. (b) Western blot analysis and densitometric quantification (underneath the blot) of the LC3-II/LC3-I ratios in NIH3T3 cells treated with rapamycin in the presence or absence of Bafilomycin A1 (100 nM). N, normal condition; R, rapamycin; R+B, rapamycin+Bafilomycin A1. (c) Schematic depiction of the dominant-negative action of UVRAGFS on the UVRAG-Beclin1 interaction by sequestering both. (d) UVRAGFS inhibits Beclin1-associated VPS34 kinase activity. NIH3T3 cells from (a) were transfected with p40(phox)-PX-GFP (to monitor phosphatidylinositol 3-phosphate formation). At 16 h post-transfection, cells were subjected to confocal microscopy and p40(phox)-PX-GFP puncta per cell were quantified. Data represent the means±s.d. (n=3). **P<0.01. Scale bar, 10 μm. (e) UVRAGFS promotes cell proliferation in Atg5-knockout iMEFs. Atg5+/+ and Atg5−/− iMEF cells stably expressing vector and UVRAGFS were seeded and counted in triplicate on day 8. Values are mean±s.d. (n=3). UVRAG and Atg5 expression was assessed by western blot with actin serving as a loading control. *P<0.05; **P<0.01. (f–h) Anchorage-independent growth of Atg5-knockout iMEFs expressing UVRAGFS. Note the larger and greater number of colonies in UVRAGFS-expressing cells. Colony diameters (g) and numbers (h) were quantified from 20 random HPFs. Data are the means±s.d. (n=3). *P<0.05; ***P<0.001. Scale bar, 50 μm.
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f3: UVRAGFS inhibits cellular autophagy in a dominant-negative manner.(a) NIH3T3 cells stably expressing vector, UVRAGWT, and UVRAGFS were transfected with GFP–LC3 and treated with rapamycin (100 nM). GFP–LC3 puncta per cell were quantified as shown in representative images shown. Data represent the means±s.d. (n=6). *P<0.05. Scale bar, 10 μm. (b) Western blot analysis and densitometric quantification (underneath the blot) of the LC3-II/LC3-I ratios in NIH3T3 cells treated with rapamycin in the presence or absence of Bafilomycin A1 (100 nM). N, normal condition; R, rapamycin; R+B, rapamycin+Bafilomycin A1. (c) Schematic depiction of the dominant-negative action of UVRAGFS on the UVRAG-Beclin1 interaction by sequestering both. (d) UVRAGFS inhibits Beclin1-associated VPS34 kinase activity. NIH3T3 cells from (a) were transfected with p40(phox)-PX-GFP (to monitor phosphatidylinositol 3-phosphate formation). At 16 h post-transfection, cells were subjected to confocal microscopy and p40(phox)-PX-GFP puncta per cell were quantified. Data represent the means±s.d. (n=3). **P<0.01. Scale bar, 10 μm. (e) UVRAGFS promotes cell proliferation in Atg5-knockout iMEFs. Atg5+/+ and Atg5−/− iMEF cells stably expressing vector and UVRAGFS were seeded and counted in triplicate on day 8. Values are mean±s.d. (n=3). UVRAG and Atg5 expression was assessed by western blot with actin serving as a loading control. *P<0.05; **P<0.01. (f–h) Anchorage-independent growth of Atg5-knockout iMEFs expressing UVRAGFS. Note the larger and greater number of colonies in UVRAGFS-expressing cells. Colony diameters (g) and numbers (h) were quantified from 20 random HPFs. Data are the means±s.d. (n=3). *P<0.05; ***P<0.001. Scale bar, 50 μm.

Mentions: UVRAGFS retains the N-terminal proline-rich and C2 domains, and the partial CCD required for Beclin1-mediated autophagy (Supplementary Fig. 1a)1012212223. To determine whether UVRAGFS retained its autophagy activity, we measured the subcellular distribution of the autophagy marker green-fluorescent protein (GFP)-LC3 and the levels of the autophagosome-associated lipidated LC3 (LC3-II)2425 in noncancerous NIH3T3 cells. As shown previously101226, UVRAGWT or rapamycin markedly promoted autophagy, as evidenced by increased GFP–LC3 puncta per cell, increased LC3-II conversion and increased response to the late-stage autophagy inhibitor Bafilomycin A1 (Fig. 3a,b). In sharp contrast, UVRAGFS did not demonstrate any proautophagic activity. Furthermore, UVRAGWT autophagy-promoting activity was abrogated when UVRAGFS was added to the cells dose dependently (Supplementary Fig. 3a). UVRAG interacts with Beclin1 through their respective CCD, resulting in activation of Beclin1-associated Vps34 kinase27. On UVRAGFS expression, the endogenous association between UVRAGWT and Beclin1 was diminished, and UVRAGFS was able to sequester the Beclin1 and UVRAG proteins in vivo, in line with its dominant-negative effect (Supplementary Fig. 3b, Fig. 3c). Accordingly, Vps34 enzymatic activity was significantly reduced in UVRAGFS cells (Fig. 3d), as illustrated by decreased punctate staining of the Vps34 kinase product, phosphatidylinositol 3-phosphate28. Impaired autophagy was also observed in vivo in NIH3T3 tumour xenografts expressing UVRAGFS (Fig. 2e), showing increased levels of p62, an autophagic substrate29. To explore whether autophagy inhibition underlies UVRAGFS-mediated oncogenesis, we examined the transforming effect of UVRAGFS in autophagy- Atg5-deficient MEFs29. UVRAGFS promoted cell proliferation (Fig. 3e) and colony growth in soft agar (Fig. 3f–h), irrespective of the autophagy status. These data support a direct role of UVRAGFS in promoting tumorigenesis independently of autophagy.


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 inhibits cellular autophagy in a dominant-negative manner.(a) NIH3T3 cells stably expressing vector, UVRAGWT, and UVRAGFS were transfected with GFP–LC3 and treated with rapamycin (100 nM). GFP–LC3 puncta per cell were quantified as shown in representative images shown. Data represent the means±s.d. (n=6). *P<0.05. Scale bar, 10 μm. (b) Western blot analysis and densitometric quantification (underneath the blot) of the LC3-II/LC3-I ratios in NIH3T3 cells treated with rapamycin in the presence or absence of Bafilomycin A1 (100 nM). N, normal condition; R, rapamycin; R+B, rapamycin+Bafilomycin A1. (c) Schematic depiction of the dominant-negative action of UVRAGFS on the UVRAG-Beclin1 interaction by sequestering both. (d) UVRAGFS inhibits Beclin1-associated VPS34 kinase activity. NIH3T3 cells from (a) were transfected with p40(phox)-PX-GFP (to monitor phosphatidylinositol 3-phosphate formation). At 16 h post-transfection, cells were subjected to confocal microscopy and p40(phox)-PX-GFP puncta per cell were quantified. Data represent the means±s.d. (n=3). **P<0.01. Scale bar, 10 μm. (e) UVRAGFS promotes cell proliferation in Atg5-knockout iMEFs. Atg5+/+ and Atg5−/− iMEF cells stably expressing vector and UVRAGFS were seeded and counted in triplicate on day 8. Values are mean±s.d. (n=3). UVRAG and Atg5 expression was assessed by western blot with actin serving as a loading control. *P<0.05; **P<0.01. (f–h) Anchorage-independent growth of Atg5-knockout iMEFs expressing UVRAGFS. Note the larger and greater number of colonies in UVRAGFS-expressing cells. Colony diameters (g) and numbers (h) were quantified from 20 random HPFs. Data are the means±s.d. (n=3). *P<0.05; ***P<0.001. Scale bar, 50 μm.
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f3: UVRAGFS inhibits cellular autophagy in a dominant-negative manner.(a) NIH3T3 cells stably expressing vector, UVRAGWT, and UVRAGFS were transfected with GFP–LC3 and treated with rapamycin (100 nM). GFP–LC3 puncta per cell were quantified as shown in representative images shown. Data represent the means±s.d. (n=6). *P<0.05. Scale bar, 10 μm. (b) Western blot analysis and densitometric quantification (underneath the blot) of the LC3-II/LC3-I ratios in NIH3T3 cells treated with rapamycin in the presence or absence of Bafilomycin A1 (100 nM). N, normal condition; R, rapamycin; R+B, rapamycin+Bafilomycin A1. (c) Schematic depiction of the dominant-negative action of UVRAGFS on the UVRAG-Beclin1 interaction by sequestering both. (d) UVRAGFS inhibits Beclin1-associated VPS34 kinase activity. NIH3T3 cells from (a) were transfected with p40(phox)-PX-GFP (to monitor phosphatidylinositol 3-phosphate formation). At 16 h post-transfection, cells were subjected to confocal microscopy and p40(phox)-PX-GFP puncta per cell were quantified. Data represent the means±s.d. (n=3). **P<0.01. Scale bar, 10 μm. (e) UVRAGFS promotes cell proliferation in Atg5-knockout iMEFs. Atg5+/+ and Atg5−/− iMEF cells stably expressing vector and UVRAGFS were seeded and counted in triplicate on day 8. Values are mean±s.d. (n=3). UVRAG and Atg5 expression was assessed by western blot with actin serving as a loading control. *P<0.05; **P<0.01. (f–h) Anchorage-independent growth of Atg5-knockout iMEFs expressing UVRAGFS. Note the larger and greater number of colonies in UVRAGFS-expressing cells. Colony diameters (g) and numbers (h) were quantified from 20 random HPFs. Data are the means±s.d. (n=3). *P<0.05; ***P<0.001. Scale bar, 50 μm.
Mentions: UVRAGFS retains the N-terminal proline-rich and C2 domains, and the partial CCD required for Beclin1-mediated autophagy (Supplementary Fig. 1a)1012212223. To determine whether UVRAGFS retained its autophagy activity, we measured the subcellular distribution of the autophagy marker green-fluorescent protein (GFP)-LC3 and the levels of the autophagosome-associated lipidated LC3 (LC3-II)2425 in noncancerous NIH3T3 cells. As shown previously101226, UVRAGWT or rapamycin markedly promoted autophagy, as evidenced by increased GFP–LC3 puncta per cell, increased LC3-II conversion and increased response to the late-stage autophagy inhibitor Bafilomycin A1 (Fig. 3a,b). In sharp contrast, UVRAGFS did not demonstrate any proautophagic activity. Furthermore, UVRAGWT autophagy-promoting activity was abrogated when UVRAGFS was added to the cells dose dependently (Supplementary Fig. 3a). UVRAG interacts with Beclin1 through their respective CCD, resulting in activation of Beclin1-associated Vps34 kinase27. On UVRAGFS expression, the endogenous association between UVRAGWT and Beclin1 was diminished, and UVRAGFS was able to sequester the Beclin1 and UVRAG proteins in vivo, in line with its dominant-negative effect (Supplementary Fig. 3b, Fig. 3c). Accordingly, Vps34 enzymatic activity was significantly reduced in UVRAGFS cells (Fig. 3d), as illustrated by decreased punctate staining of the Vps34 kinase product, phosphatidylinositol 3-phosphate28. Impaired autophagy was also observed in vivo in NIH3T3 tumour xenografts expressing UVRAGFS (Fig. 2e), showing increased levels of p62, an autophagic substrate29. To explore whether autophagy inhibition underlies UVRAGFS-mediated oncogenesis, we examined the transforming effect of UVRAGFS in autophagy- Atg5-deficient MEFs29. UVRAGFS promoted cell proliferation (Fig. 3e) and colony growth in soft agar (Fig. 3f–h), irrespective of the autophagy status. These data support a direct role of UVRAGFS in promoting tumorigenesis independently of autophagy.

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