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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 activates Rac-1 and promotes tumour metastasis in vitro and vivo.(a) Rac-1 activation by UVRAGFS. Western blot shows a pull-down experiment to detect GTP-bound Rac1 in SW480.UVRAGFS cells and on drug treatment. Histogram shows quantification from three independent experiments. (b) Representative images of scratch-wound healing exhibit the motility of SW480.UVRAGFS cells. Cell motility into the wound area was taken at 0 and 16 h as marked by red lines. Wound-healing index was quantified (right). Data are the means±s.d. (n=3). **P<0.01; ***P<0.001; NS, not significant. Histogram shows quantification from three independent experiments. (c,d) UVRAGFS enhances tumour metastasis in mice inoculated by intrasplenic injection of SW480.Vector and SW480.UVRAGFS cells. Schematic depiction of the procedure (c, left) and representative images (c, right) of upper abdominal organs at 8-week post-injection are shown (c). The number of metastatic nodules (liver and lung) was quantified (d). H&E staining was performed on serial sections of metastatic tumours (M) and normal (N) liver and lung are shown below. Scale bar, 1 mm. Arrows in c represent the metastasis foci. Results are representative of 10 mice per group. Fisher's exact test was used. (e) Immunohistochemistry analysis of autophagy, apoptosis and EMT status of primary tumours (left panel) and metastasis nodules (right panel). The bar graphs represent the quantification of the indicated protein markers. Data are the means±s.d. (n=3). *P<0.05. Scale bar, 50 μm. (f) Western blot of the EMT-related protein expression in SW480 cells expressing UVRAGFS and on Taxol treatment.
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f5: UVRAGFS activates Rac-1 and promotes tumour metastasis in vitro and vivo.(a) Rac-1 activation by UVRAGFS. Western blot shows a pull-down experiment to detect GTP-bound Rac1 in SW480.UVRAGFS cells and on drug treatment. Histogram shows quantification from three independent experiments. (b) Representative images of scratch-wound healing exhibit the motility of SW480.UVRAGFS cells. Cell motility into the wound area was taken at 0 and 16 h as marked by red lines. Wound-healing index was quantified (right). Data are the means±s.d. (n=3). **P<0.01; ***P<0.001; NS, not significant. Histogram shows quantification from three independent experiments. (c,d) UVRAGFS enhances tumour metastasis in mice inoculated by intrasplenic injection of SW480.Vector and SW480.UVRAGFS cells. Schematic depiction of the procedure (c, left) and representative images (c, right) of upper abdominal organs at 8-week post-injection are shown (c). The number of metastatic nodules (liver and lung) was quantified (d). H&E staining was performed on serial sections of metastatic tumours (M) and normal (N) liver and lung are shown below. Scale bar, 1 mm. Arrows in c represent the metastasis foci. Results are representative of 10 mice per group. Fisher's exact test was used. (e) Immunohistochemistry analysis of autophagy, apoptosis and EMT status of primary tumours (left panel) and metastasis nodules (right panel). The bar graphs represent the quantification of the indicated protein markers. Data are the means±s.d. (n=3). *P<0.05. Scale bar, 50 μm. (f) Western blot of the EMT-related protein expression in SW480 cells expressing UVRAGFS and on Taxol treatment.

Mentions: Centrosome amplification per se has been shown to promote cell invasion through inappropriate microtubule nucleation and Rac-1 activation31, a small GTPase important for the control of cell invasiveness and metastasis3233. Indeed, pull-down assay in UVRAGFS SW480 cells detected a more than twofold Rac1 activation, which could be blocked by Taxol, but not by the autophagy inhibitor chloroquine or the anticancer reagent 5-FU (Fig. 5a), indicating a requirement for dynamic microtubules. Consistent with increased Rac1 activation, UVRAGFS enhanced the cell motility of SW480 cells in a wound-healing assay, which was inhibited by Taxol (Fig. 5b). It also enhanced HCT116 cell migration through a collagen matrix, whereas UVRAGWT exerted an inhibitory effect (Supplementary Fig. 6a,b). Spleen injection of non-metastatic SW480 cells expressing UVRAGFS into nude mice resulted in a higher incidence of liver metastasis and a greater number of colonization in the lungs, kidney and peritoneum, whereas no colonization was found in the control group (Fig. 5c,d, Supplementary Fig. 6c). UVRAGFS-induced tumour metastases were confirmed in an independent mouse metastasis model with SW480 cells expressing GFP–UVRAGFS, as determined by bioluminescence imaging of metastatic lesions (Supplementary Fig. 6d). These results indicate that UVRAGFS enhances the metastatic capacity of CRC cells.


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 activates Rac-1 and promotes tumour metastasis in vitro and vivo.(a) Rac-1 activation by UVRAGFS. Western blot shows a pull-down experiment to detect GTP-bound Rac1 in SW480.UVRAGFS cells and on drug treatment. Histogram shows quantification from three independent experiments. (b) Representative images of scratch-wound healing exhibit the motility of SW480.UVRAGFS cells. Cell motility into the wound area was taken at 0 and 16 h as marked by red lines. Wound-healing index was quantified (right). Data are the means±s.d. (n=3). **P<0.01; ***P<0.001; NS, not significant. Histogram shows quantification from three independent experiments. (c,d) UVRAGFS enhances tumour metastasis in mice inoculated by intrasplenic injection of SW480.Vector and SW480.UVRAGFS cells. Schematic depiction of the procedure (c, left) and representative images (c, right) of upper abdominal organs at 8-week post-injection are shown (c). The number of metastatic nodules (liver and lung) was quantified (d). H&E staining was performed on serial sections of metastatic tumours (M) and normal (N) liver and lung are shown below. Scale bar, 1 mm. Arrows in c represent the metastasis foci. Results are representative of 10 mice per group. Fisher's exact test was used. (e) Immunohistochemistry analysis of autophagy, apoptosis and EMT status of primary tumours (left panel) and metastasis nodules (right panel). The bar graphs represent the quantification of the indicated protein markers. Data are the means±s.d. (n=3). *P<0.05. Scale bar, 50 μm. (f) Western blot of the EMT-related protein expression in SW480 cells expressing UVRAGFS and on Taxol treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526116&req=5

f5: UVRAGFS activates Rac-1 and promotes tumour metastasis in vitro and vivo.(a) Rac-1 activation by UVRAGFS. Western blot shows a pull-down experiment to detect GTP-bound Rac1 in SW480.UVRAGFS cells and on drug treatment. Histogram shows quantification from three independent experiments. (b) Representative images of scratch-wound healing exhibit the motility of SW480.UVRAGFS cells. Cell motility into the wound area was taken at 0 and 16 h as marked by red lines. Wound-healing index was quantified (right). Data are the means±s.d. (n=3). **P<0.01; ***P<0.001; NS, not significant. Histogram shows quantification from three independent experiments. (c,d) UVRAGFS enhances tumour metastasis in mice inoculated by intrasplenic injection of SW480.Vector and SW480.UVRAGFS cells. Schematic depiction of the procedure (c, left) and representative images (c, right) of upper abdominal organs at 8-week post-injection are shown (c). The number of metastatic nodules (liver and lung) was quantified (d). H&E staining was performed on serial sections of metastatic tumours (M) and normal (N) liver and lung are shown below. Scale bar, 1 mm. Arrows in c represent the metastasis foci. Results are representative of 10 mice per group. Fisher's exact test was used. (e) Immunohistochemistry analysis of autophagy, apoptosis and EMT status of primary tumours (left panel) and metastasis nodules (right panel). The bar graphs represent the quantification of the indicated protein markers. Data are the means±s.d. (n=3). *P<0.05. Scale bar, 50 μm. (f) Western blot of the EMT-related protein expression in SW480 cells expressing UVRAGFS and on Taxol treatment.
Mentions: Centrosome amplification per se has been shown to promote cell invasion through inappropriate microtubule nucleation and Rac-1 activation31, a small GTPase important for the control of cell invasiveness and metastasis3233. Indeed, pull-down assay in UVRAGFS SW480 cells detected a more than twofold Rac1 activation, which could be blocked by Taxol, but not by the autophagy inhibitor chloroquine or the anticancer reagent 5-FU (Fig. 5a), indicating a requirement for dynamic microtubules. Consistent with increased Rac1 activation, UVRAGFS enhanced the cell motility of SW480 cells in a wound-healing assay, which was inhibited by Taxol (Fig. 5b). It also enhanced HCT116 cell migration through a collagen matrix, whereas UVRAGWT exerted an inhibitory effect (Supplementary Fig. 6a,b). Spleen injection of non-metastatic SW480 cells expressing UVRAGFS into nude mice resulted in a higher incidence of liver metastasis and a greater number of colonization in the lungs, kidney and peritoneum, whereas no colonization was found in the control group (Fig. 5c,d, Supplementary Fig. 6c). UVRAGFS-induced tumour metastases were confirmed in an independent mouse metastasis model with SW480 cells expressing GFP–UVRAGFS, as determined by bioluminescence imaging of metastatic lesions (Supplementary Fig. 6d). These results indicate that UVRAGFS enhances the metastatic capacity of CRC cells.

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