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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

Cell transformation and oncogenic effect of UVRAGFS mutant.(a) NIH3T3 cells stably expressing empty vector, UVRAGWT, and UVRAGFS (104) were seeded and counted over time in triplicate. Values are the means±s.d. (n=4). Flag-tagged UVRAG expression is shown by western blot and β-tubulin serves as a loading control. *P<0.05; **P<0.01. (b) NIH3T3 cells described above were plated at low density (2,500 cells per 10-cm plate), grown for 14 days then fixed and stained with crystal violet. (c) Anchorage-independent growth induced by UVRAGFS. NIH3T3 cells in (a) were seeded in 0.3% top agar and incubated for 20 days. UVRAGFS-expressing cells formed larger and greater number of colonies in soft agar. Representative images of colonies are shown and the quantitative results of colony numbers were obtained from 10 randomly chosen HPF. Data represent the means±s.d. (n=4). **P<0.01; ****P<0.0001. Scale bar, 500 μm. (d) UVRAGFS-associated oncogenesis in nude mouse model. UVRAGFS-NIH3T3 cells from (a) were subcutaneously injected into flanks of nude mice, and tumour growth was measured over time. Circles indicate xenograft tumours at day 38 after inoculation. Data shown are representative of three separate experiments. (e) Immunohistochemistry staining of 3T3-tumours with the indicated antibodies and their quantification in bar graphs (bottom). Arrows denote the mitotic and Ki67+ proliferating cells in the tumour. **P<0.01. Scale bar, 50 μm.
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f2: Cell transformation and oncogenic effect of UVRAGFS mutant.(a) NIH3T3 cells stably expressing empty vector, UVRAGWT, and UVRAGFS (104) were seeded and counted over time in triplicate. Values are the means±s.d. (n=4). Flag-tagged UVRAG expression is shown by western blot and β-tubulin serves as a loading control. *P<0.05; **P<0.01. (b) NIH3T3 cells described above were plated at low density (2,500 cells per 10-cm plate), grown for 14 days then fixed and stained with crystal violet. (c) Anchorage-independent growth induced by UVRAGFS. NIH3T3 cells in (a) were seeded in 0.3% top agar and incubated for 20 days. UVRAGFS-expressing cells formed larger and greater number of colonies in soft agar. Representative images of colonies are shown and the quantitative results of colony numbers were obtained from 10 randomly chosen HPF. Data represent the means±s.d. (n=4). **P<0.01; ****P<0.0001. Scale bar, 500 μm. (d) UVRAGFS-associated oncogenesis in nude mouse model. UVRAGFS-NIH3T3 cells from (a) were subcutaneously injected into flanks of nude mice, and tumour growth was measured over time. Circles indicate xenograft tumours at day 38 after inoculation. Data shown are representative of three separate experiments. (e) Immunohistochemistry staining of 3T3-tumours with the indicated antibodies and their quantification in bar graphs (bottom). Arrows denote the mitotic and Ki67+ proliferating cells in the tumour. **P<0.01. Scale bar, 50 μm.

Mentions: To probe whether the UVRAGFS mutant represents a mere loss of WT function11 as occurs with most other tumour suppressors, or imparts oncogenic properties, we established MSS SW480 and MSI HCT116 cell lines stably expressing Flag-tagged UVRAGWT and UVRAGFS at equivalent levels (Supplementary Fig. 2a,d). UVRAGFS-transduced cells showed increased proliferation and enhanced anchorage-independent growth in soft agar (Supplementary Fig. 2a–e), independently of the tissue of origin (Supplementary Fig. 2f,g). Subcutaneous transplantation in athymic nude mice of UVRAGFS SW480 cells resulted in tumour formation with accelerated kinetics (Supplementary Fig. 2c). To further test whether expression of UVRAGFS is sufficient to transform noncancerous cells, we used NIH3T3 mouse embryonic fibroblasts stably expressing UVRAGWT or UVRAGFS (Fig. 2a). Compared with control (3T3.Vec), UVRAGFS–3T3 cells had elevated growth rate, formed larger colonies when plated at low density and induced anchorage-independent growth, whereas UVRAGWT exerted the opposite effects (Fig. 2a–c). The tumour growth rate and mean tumour volume were drastically increased when 3T3–UVRAGFS cells were injected into nude mice (Fig. 2d). Immunohistological analyses of tumour xenografts showed UVRAGFS expression and enhanced mitotic index and number of Ki67+ (proliferating) cells in UVRAGFS tumours (Fig. 2e). CRC primary tumours with the FS mutation also had increased Ki67 staining (Fig. 1d). Altogether, these data indicate a strong association of the cancer-derived UVRAGFS with a tumorigenic phenotype.


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)

Cell transformation and oncogenic effect of UVRAGFS mutant.(a) NIH3T3 cells stably expressing empty vector, UVRAGWT, and UVRAGFS (104) were seeded and counted over time in triplicate. Values are the means±s.d. (n=4). Flag-tagged UVRAG expression is shown by western blot and β-tubulin serves as a loading control. *P<0.05; **P<0.01. (b) NIH3T3 cells described above were plated at low density (2,500 cells per 10-cm plate), grown for 14 days then fixed and stained with crystal violet. (c) Anchorage-independent growth induced by UVRAGFS. NIH3T3 cells in (a) were seeded in 0.3% top agar and incubated for 20 days. UVRAGFS-expressing cells formed larger and greater number of colonies in soft agar. Representative images of colonies are shown and the quantitative results of colony numbers were obtained from 10 randomly chosen HPF. Data represent the means±s.d. (n=4). **P<0.01; ****P<0.0001. Scale bar, 500 μm. (d) UVRAGFS-associated oncogenesis in nude mouse model. UVRAGFS-NIH3T3 cells from (a) were subcutaneously injected into flanks of nude mice, and tumour growth was measured over time. Circles indicate xenograft tumours at day 38 after inoculation. Data shown are representative of three separate experiments. (e) Immunohistochemistry staining of 3T3-tumours with the indicated antibodies and their quantification in bar graphs (bottom). Arrows denote the mitotic and Ki67+ proliferating cells in the tumour. **P<0.01. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526116&req=5

f2: Cell transformation and oncogenic effect of UVRAGFS mutant.(a) NIH3T3 cells stably expressing empty vector, UVRAGWT, and UVRAGFS (104) were seeded and counted over time in triplicate. Values are the means±s.d. (n=4). Flag-tagged UVRAG expression is shown by western blot and β-tubulin serves as a loading control. *P<0.05; **P<0.01. (b) NIH3T3 cells described above were plated at low density (2,500 cells per 10-cm plate), grown for 14 days then fixed and stained with crystal violet. (c) Anchorage-independent growth induced by UVRAGFS. NIH3T3 cells in (a) were seeded in 0.3% top agar and incubated for 20 days. UVRAGFS-expressing cells formed larger and greater number of colonies in soft agar. Representative images of colonies are shown and the quantitative results of colony numbers were obtained from 10 randomly chosen HPF. Data represent the means±s.d. (n=4). **P<0.01; ****P<0.0001. Scale bar, 500 μm. (d) UVRAGFS-associated oncogenesis in nude mouse model. UVRAGFS-NIH3T3 cells from (a) were subcutaneously injected into flanks of nude mice, and tumour growth was measured over time. Circles indicate xenograft tumours at day 38 after inoculation. Data shown are representative of three separate experiments. (e) Immunohistochemistry staining of 3T3-tumours with the indicated antibodies and their quantification in bar graphs (bottom). Arrows denote the mitotic and Ki67+ proliferating cells in the tumour. **P<0.01. Scale bar, 50 μm.
Mentions: To probe whether the UVRAGFS mutant represents a mere loss of WT function11 as occurs with most other tumour suppressors, or imparts oncogenic properties, we established MSS SW480 and MSI HCT116 cell lines stably expressing Flag-tagged UVRAGWT and UVRAGFS at equivalent levels (Supplementary Fig. 2a,d). UVRAGFS-transduced cells showed increased proliferation and enhanced anchorage-independent growth in soft agar (Supplementary Fig. 2a–e), independently of the tissue of origin (Supplementary Fig. 2f,g). Subcutaneous transplantation in athymic nude mice of UVRAGFS SW480 cells resulted in tumour formation with accelerated kinetics (Supplementary Fig. 2c). To further test whether expression of UVRAGFS is sufficient to transform noncancerous cells, we used NIH3T3 mouse embryonic fibroblasts stably expressing UVRAGWT or UVRAGFS (Fig. 2a). Compared with control (3T3.Vec), UVRAGFS–3T3 cells had elevated growth rate, formed larger colonies when plated at low density and induced anchorage-independent growth, whereas UVRAGWT exerted the opposite effects (Fig. 2a–c). The tumour growth rate and mean tumour volume were drastically increased when 3T3–UVRAGFS cells were injected into nude mice (Fig. 2d). Immunohistological analyses of tumour xenografts showed UVRAGFS expression and enhanced mitotic index and number of Ki67+ (proliferating) cells in UVRAGFS tumours (Fig. 2e). CRC primary tumours with the FS mutation also had increased Ki67 staining (Fig. 1d). Altogether, these data indicate a strong association of the cancer-derived UVRAGFS with a tumorigenic phenotype.

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